Method for mycoplasma detection in a biological sample

ABSTRACT

The present invention provides a method for detecting mycoplasma in a biological sample through the application of nucleic acid hybridization techniques. More specifically, the instant invention details a method of detecting a wide variety of mycoplasma in a biological sample by employing a polynucleotide segment encoding a portion of M. pneumoniae P1 polypeptide.

The Government may own certain rights in this invention pursuant toNational Institute of Health, Grant Number AI 18540, awarded by theDepartment of Health & Human Sciences.

This application is a continuation of application Ser. No. 07/558,886,filed Jul. 27, 1990, abandoned, which is a continuation-in-part ofapplication Ser. 07/118,967, filed Nov. 10, 1987, U.S. Pat. No.5,026,636, which is a continuation-in-part of application Ser. No.07/004,767, filed Jan. 9, 1987, U.S. Pat. No. 4,945,041.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the molecular cloning of the gene encodingMycoplasma pneumoniae P1 cytadhesin protein and to related mycoplasmalgenes and proteins, e.g., from Mycoplasma genitalium, Mycoplasmagallisepticum, Mycoplasma incognitus, Mycoplasma fermentans, Mycoplasmasualvi, Mycoplasma hominis, and Mycoplasma pulmonis.

The P1 protein of Mycoplasma pneumoniae mediates mycoplasmalcolonization of host respiratory epithelium and is a critical virulencedeterminant. By the present invention, a complete DNA sequence of thecomplete P1 gene as well as a deduced amino acid sequence of the P1cytadhesin protein is presented for the first time. In addition, clonesexpressing M. pneumoniae peptides are provided. These peptides containfunctional epitopes and have been used to localize the cytadhesinbinding domain of P1.

More particularly, this invention describes using specific P1 adhesingene sequences and peptides screening mycoplasmal pathogens in differentspecies. This invention also describes using the P1 specific peptidesfor the production of a vaccine for use as a preventive means in speciesthat potentially are plagued with mycoplasmal infections.

2. Description of the Related Art

In humans, mycoplasma-associated diseases present a wide spectrum ofclinical symptoms ranging from acute respiratory illness withextrapulmonary manifestations such as central nervous system involvementto genitourinary tract and joint infections. In animals, mycoplasmascause well-characterized respiratory and arthritic disorders. In insectsand plants pathogenic mycoplasmas (spiroplasmas) demonstrate a broadhost range specificity. It appears that mycoplasmas as a group areextraordinary pathogens capable of invasive or chronic disease indiverse hosts, producing a variety of clinical manifestations andfrequently capable of suppressing host-defense mechanisms. Diagnosticreagents and vaccines are needed in order to effectively diagnose andprevent mycoplasmal infection.

Mycloplasmas

Mycoplasmas, prokaryotic pathogens, are now designated class Mollicutes,with three families and four genera. Mycoplasmas are resistant topenicillin and antibiotics known to interfere with polymerization ofcell wall precursors. They are inhibited by tetracyclines and inselected instances, erythromycin. Like bacteria, they grow outside thecell, possess ribonucleic and deoxyribonucleic acids and reproduce byfission.

A. Mycoplasma pneumoniae

The mycoplasma of chief importance in human disease is Mycoplasmapneumoniae, a respiratory pathogen. M. pneumoniae is a non-invasivepathogen that colonizes the mucosal surface of the respiratory tract andcauses a primary, atypical pneumonia. Pneumonia caused by M. pneumoniaeis characterized by fever, pharyngitis, cough and pulmonary infection.This bacterial organism can also cause upper respiratory illness withoutpneumonia and asymptomatic infection. Although this disease appears tooccur most frequently in young adults and children, its incidence in thegeneral population may be underestimated because the symptoms are oftenrelatively mild and diagnostic procedures are suboptimal.

M. pneumoniae initiates infection by colonizing cells of the respiratoryepithelium. This colonization is mediated by a specialized tip-likeorganelle containing clusters of a surface-localized, trypsin sensitiveprotein designated P1. Numerous studies show P1 to be a criticalvirulence determinant. For example, mutants of M. pneumoniae that lackP1 or are unable to mobilize and anchor P1 at the tip are avirulent. Inaddition, treatment of virulent M. pneumoniae with trypsin abrogatesadherence to the respiratory epithelium. Finally, monoclonal antibodiesto P1 have been shown to block M. pneumoniae cytadherence. Plummer, etal., Infect. Immun., 53:398-403 (1986).

Unfortunately, despite the critical importance of P1 as a mycoplasmalvirulence determinant, efforts to provide a cloned gene encoding the P1cytadhesin have been generally unsatisfactory. For example, Trevino, etal., Infect. Immun., 53:129-134 (1986), describe an attempt to clone M.pneumoniae antigens by constructing an M. pneumoniae genomic libraryusing lambda phage EMBL3 as the vector and immunoscreening the librarywith adsorbed anti-M. pneumoniae serum. Although this procedure producedseveral clones exhibiting antigenic cross-reactivity with M. pneumoniaeP1, none of the clones reacted with monoclonal antibodies specific forcritical antigenic determinants of P1 shown by the present inventors tomediate cytadherence. Moreover, the largest immunoreactive proteinidentified had a molecular weight of only 140 kDa. In contrast, nativeP1 has a molecular weight of approximately 165 kDa. Therefore, it couldnot be definitely established whether or not the 140 kDa protein was aproduct of the structural P1 gene. The approach was then abandoned.

Since the P1 cytadhesin is probably the most important mediator ofmycoplasma cytadsorption, further elucidation of the structure of thismolecule is likely to provide information essential for a completeunderstanding of the role of cytadherence in pathogenesis of mycoplasmaldisease. This goal can be achieved most readily by cloning andsequencing the structural gene encoding P1. Furthermore, recent studieshave shown that adherence of mycoplasma to respiratory epithelium can beinhibited by certain antibodies directed against cytadhesin epitopes ofP1. Therefore, vaccines comprising recombinant P1 protein or selectedcytadhesin polypeptides derived from recombinant P1 are likely to proveeffective in preventing mycoplasmal infection. In addition, theavailability of the complete gene sequence and deduced amino acidsequence for M. pneumoniae P1 will allow one to map critical antigenicepitopes and produce selected synthetic peptides useful as diagnosticprobes or vaccines.

B. Mycoplasma genitalium

M. genitalium is currently under investigation as the possible cause ofone sexually transmitted disease, nongonococcal urethritis. About 40% ofnongonococcal urethritis is caused by Chlamydia trachomatis. A smallproportion of these cases are caused by Herpes simplex virus orTrichomonas vaginalis, while about 50% of cases cannot be specificallyattributed to any of these pathogens except possibly M. genitalium.

Like Mycoplasma pneumoniae, M. genitalium penetrates host defensebarriers and parasitize tissues via a unique differentiated tiporganelles that exhibits a nap-like appearance. Earlier reportsindicated that the adhesin proteins of M. pneumoniae and M. genitaliumcluster at the tip structures and regulate attachment and recognition ofhost receptors [Baseman, J. B., et al., J. Bacteriol. 151:1514-1522(1982)]. In M. genitalium, a 140 kDa adhesin that shares cross reactiveepitopes with the P1 adhesin of M. pneumoniae has been identified [W. A.Clyde Jr., et al. Infect. Immun. 51:690-692 (1986)].

Also, DNA and protein sequence homologies between the cytadhesins of M.pneumoniae and M. genitalium have been described. The homology isnoteworthy because the P1 gene of M. pneumoniae has an A+T content of46.5% while the M. genitalium adhesin gene has an A+T content of 60.1%,consistent with the preferential use of A- and T-rich codons by M.genitalium [S. F. Dallo et al., Microb. Patho. 6:69-73 (1989); S. F.Dallo et al., Infect. Immun. 57:1059-1065 (1989)]. Considerabledifferences in the G+C contents of genomic DNA of M. pneumoniae and M.genitalium exist as well.

C. Mycoplasma incognitus

Recently, a new mycoplasma, Mycoplasma incognitus was detected in tissueof patients with Acquired Immune Deficiency Syndrome (AIDS). S. C. Lo etal., Am. J. Trop. Med. Hyg. 41(5):586-600 (1989). Mycoplasma incognituswere first detected in the autopsy tissues and peripheral bloodmononuclear cells of homosexuals, drug abusers andtransfusion-associated patients with AIDS using the polymerass chainreaction (PCR) technique, immunocytochemistry and electron microscopy.Numerous particles with mycoplasma morphology were visualizedextracellularly and intracellularly in specific tissues wherehistopathology ranged from minimal to extensive necrosis, with orwithout evidence of an inflammatory response. No other microbial agentswere found in these lesions. Therefore, a pathogenic role for Mycoplasmaincognitus was suggested, although it remained unclear whether M.incognitus is an opportunistic pathogen, a cofactor, or a primary causeof the pathology observed. There is data to support the hypothesis thatM. incognitus is infectious and responsible for AIDS progression. S. C.Lo et al., Am. J. Trop. Med. Hyg. 40(2):213-226 (1989); S. C. Lo et al.,Am, J. Trop. Med. Hyg. 40(4):399-400 (1989); S. C. Lo et al., Am. J.Trop. Med. Hyg. 41(5):601-616 (1989).

M. incognitus was also implicated in fulminant infections of 6geographically unrelated, previously healthy non-AIDS patients whopresented with acute flu-like syndromes and died with multi-tissuenecrosis in 1 to 7 weeks after the onset of symptoms. No otheretiological agent was identified in these non-AIDS patients. A minimalcellular immune response with few inflammatory cells was observed inthese same tissues suggesting that M. incognitus may possessimmunosuppressive properties or share antigens with the host, therebyavoiding cell-mediated immune defenses.

Additional studies in experimentally infected monkeys have directlyestablished that M. incognitus can cause systemic disease. M. incognituswas purified from Kaposi's sarcoma DNA-transfected NIH/3T3 cells andinjected intraperitoneally into an experimental animal model systeminvolving silvered leaf monkeys. All test animals developed systemicinfections, displayed poor antibody responses with no acute inflammatorylesions, exhibited wasting syndromes and died within 7 to 9 months. Apoor inflammatory reactive process accompanied by a weak antibodyresponse was observed in all animals. Upon autopsy, M. incognits wasfound in the cytoplasm and nuclei of necropsy tissues, as well asextracellularly; no evidence of other causative agents was found.

M. incognitus, which utilizes glucose aerobically and anaerobically andmetabolizes arginine, is similar to Mycoplasma fermentans in somerespects. Immunologic cross-reactivity and similar patterns ofantibiotic resistance between the two species have been reported.Another shared chatacteristic between M. incognitus and M. fermentansincludes the ability to transform eukaryotic cells in culture. Inaddition, M. fermentans, which was first identified as a humanurogenital isolate in 1950, has been isolated from the genital tracts ofapparently healthy individuals, the bone marrow of leukemic patients,and as a tissue culture contaminant. M. fermentans has also been shownto cause various pathologies in experimentally infected mice andmonkeys. However, monoclonal antibodies generated againstheat-inactivated M. incognitus distinguish M. incognitus from M.fermentans. Also, M. incognitus colony morphology, size of individualmycoplasmas and growth kinetics differ from known M. fermentans strains.

D. Mycoplasma gallisepticum

Mycoplasma gallisepticum is an avian pathogen [Baseman, J. B., Banai M.,Kahane I., Infect. Immun. 38:389-391 (1982)]. To date, the existence ofan adhesin protein in an avian pathogen, M. gallisepticum, has been lessclear, although evidence suggests that a common population of receptorson red blood cells mediate adherence of these mycoplasma species [J. B.Baseman et al., Infect. Immun. 43:1103-1105 (1984); W. A. Clyde et al.,Infect. Immun. 51:690-692 (1986)].

E. Other Mycoplasmas

Other mycloplasmas, such as M. hominus, M. fermentans, M. sualvi (a pigpathogen), and M. pulmonis (a rodent pathogen) have also been postulatedto play important roles in pathogenesis of man and animals. From thediscussion above, one can appreciate that there is a need to identifycommon threads between these and other species of mycoplasma. Thisinvention provides the identity of a number of common epitopes, whichprovide the basis for a development of vaccines and diagnostic reagentsbased on shared epitopes of mycoplasmal adhesins.

SUMMARY OF THE INVENTION

By the present invention, the cloning and DNA sequencing of the completeP1 gene is described for the first time. In addition, the complete aminosequence of the P1 protein is provided. The invention also providesrecombinant P1 polypeptides, including polypeptides expressed as fusionproteins comprising cytadhesin epitopes. Accordingly, in a general andoverall scope, the present invention comprises recombinant clonesencoding P1, recombinant DNA sequences suitable for use as hybridizationprobes to assist cloning of genes encoding P1 and other mycoplasmalcytadhesins, methods for isolating such genes, and recombinant P1polypeptides.

More particularly, the invention relates to substantially purifiednucleic acid molecules comprising a nucleotide sequence encoding the P1protein or portion of the C-terminal portion thereof. Of course,absolute purification of the nucleic acid molecule is not necessary.Rather, the term "substantially purified" is intended to distinguish theclaimed species from species found in nature. Moreover, it will beappreciated that there is no requirement that the nucleic acid encode acomplete P1 protein. All that is required is that the molecule encode atleast a portion of the C-terminal portion of the P1 protein. For thepurposes of the present invention, a C-terminal portion of P1 is definedas the portion of P1 encoded by nucleotides downstream from nucleotide2440.

In a further embodiment, the substantially purified nucleic acidmolecule encodes a P1 protein having molecular weight of about 165-170kDa. In yet still a further embodiment, the invention relates to anucleic acid molecule wherein the nucleotide sequence is defined as anucleotide sequence encoding the amino acid sequence of FIG. 6. Althoughthe term nucleic acid is meant to include both ribonucleic acid (RNA)and deoxyribonucleic acid (DNA), DNA is preferred for the purposes ofthe present invention. Accordingly, in one embodiment, the nucleic acidis described as DNA.

In addition, the invention provides a substantially purified nucleicacid molecule comprising a nucleotide sequence encoding an M. pneumoniaeP1 polypeptide having a cytadhesin epitope. For purposes of the presentinvention, a polypeptide is defined as a peptide of more than one aminoacid, and a P1 cytadhesin epitope is considered to be any P1 polypeptidewhich binds to an antibody capable of inhibiting P1 mediatedcytadherence or is itself capable of competitively inhibiting P1mediated cytadherence. For example, a more specific embodiment relatesto a nucleic acid molecule wherein the cytadhesin epitope encoded iscapable of reacting immunologically with monoclonal antibody 5B8,produced by ATCC# HB 5986.

Similarly, an additional embodiment is directed toward a nucleic acidmolecule where the cytadhesin peptide is capable of reactingimmunologically with monoclonal antibody 6E7, produced by ATCC# HB 8420.Further embodiments of the invention relate to nucleic acid moleculescomprising DNA sequences encoding M. pneumoniae P1 polypeptides of atleast thirteen amino acids in length. More specifically, the inventionprovides for nucleic acid molecules wherein the P1 polypeptide comprisesat least the particular amino acid sequence of the thirteen amino acidcytadhesin epitope described by the present inventors, or the amino acidsequences corresponding to those expressed by phage clones P1-7, P1-9,and P1-10.

Of course, as those of skill in the art will appreciate, the DNAsequences claimed are not required to be actively expressing mycoplasmalP1 polypeptides or in a proper expression vector or expression frame toexpress the polypeptide. In addition, due to the redundancy of thegenetic code, a number of nucleotide sequences may encode the indicatedamino acid sequences. Any such nucleotide sequence is considered to bewithin the scope of the present invention. Moreover, those of skill inthe art will appreciate that, in some cases, conservative amino acidsubstitutions may allow the production of a polypeptide which has aslightly different amino acid sequence than any of those recited in theclaims, but has essentially identical function. Such polypeptides areconsidered to be functional equivalents of the polypeptides describedherein and nucleotide sequences encoding such polypeptides areconsidered to be within the scope of the present claims.

Additional embodiments of the invention are directed towards DNA vectorscomprising any one of the DNA molecules described above as well astoward bacterial strains comprising such recombinant vectors. In a moreparticular embodiment, the bacterial strain is defined as E. coli.

A further embodiment of the invention is directed towards DNA moleculescomprising a DNA sequence which includes at least a tetradecamericportion of the DNA sequence of FIG. 6. These DNA molecules are believedto possess a number of utilities. First, they may be used ashybridization probes to assist in cloning the P1 gene from M.pneumoniae. In addition, it is contemplated that they may be used todetect homologous nucleotide sequences in other mycoplasmal species, andthus aid in cloning of cytadhesin genes from such species, M.genitalium, for example. Of course, in some cases they may also be usedto direct synthesis of the polypeptides encoded.

In more specific embodiments of this aspect of the invention, particularnucleotides sequences are claimed. For example, embodiments directed toDNA molecules wherein the DNA sequence comprises nucleotides 178 to 192or 196 to 213 of FIG. 6 are essentially directed to the 14-mer and18-mer probes encoding the amino acids shown in FIGS. 2A or 2B,respectively. A further embodiment, directed towards the DNA sequencecomprising nucleotides -70 to 258 of the DNA sequence of FIG. 6 isessentially directed towards the nucleotide sequence of the Hae IIIrestriction fragment described herein. A still further embodiment isdirected to the EcoRI/Pst I restriction fragment (nucleotides -204 to911) used by the present inventors to isolate the P1 gene. Of course,yet another embodiment is directed to the DNA fragment containing thecomplete structural gene itself. Additional embodiments are directed tothe DNA molecule encoding the thirteen amino acid cytadhesin epitope(nucleotides 4148-4185) and to DNA sequences comprising the DNA insertsof phage clones P1-7 (nucleotides 4067-4085), P1-9 (nucleotides4148-4881), and P1-10 (nucleotides 4202-4881), respectively.

Yet still a further embodiment is directed to a DNA sequence comprisingat least the transcribed portion of the DNA sequence of FIG. 6. Yetstill a further embodiment is directed to the DNA sequence of FIG. 6.

It will be readily apparent to those skilled in the art that FIG. 6depicts both the coding and non-coding strand of the P1 DNA. It shouldbe expressly pointed out that the claims of the present invention arenot limited to double stranded DNA molecules, but are intended toencompass both double stranded and single stranded molecules, since, inmany systems, either strand may be used as a nucleotide probe and onestrand may easily be produced from its complementary strand. The onlyrequirement is that the DNA molecule include the specified nucleotidesequence.

In addition, although certain embodiments refer only to DNA molecules,those of skill in the art will also appreciate that the DNA sequences ofthe present invention can easily be transcribed into a corresponding RNAmolecule. Therefore, RNA molecules corresponding to the DNA sequences ofthe present invention are considered to be functional equivalents ofsuch DNA molecules and are intended to be encompassed by the presentclaims.

Additional embodiments of the invention relate to DNA molecules capableof hybridizing to the recombinant insert of the 6 kbp EcoRI fragmentdesignated plasmid pMPN P1 under selected hybridization conditions, saidmolecules suitable for use as hybridization probes. For example, oneembodiment is directed toward a DNA molecule capable of hybridizing tothe recombinant insert of plasmid pMPN P1, obtainable from ATCC# 67560under moderately stringent hybridization conditions while anotherembodiment is directed toward a DNA molecule capable of hybridizing tothe recombinant insert of plasmid pMPN P1, obtainable from ATCC# 67560under stringent hybridization conditions. For the purposes of thepresent invention, such conditions are described as moderately stringentin that they allow detection of a nucleotide sequence at least 14nucleotides in length having at least approximately 75% homology withthe sequence of the nucleotide probe used. Stringent hybridizationconditions are defined as conditions wherein the probe detectsnucleotide sequences at least 14 nucleotides in length having a homologygreater than about 90%. The conditions necessary for hybridization of aparticular probe to a particular nucleotide sequence having a specifieddegree of homology may be determined by referring to Nucleic AcidHybridization, A Practical Approach, Hames and Higgins, eds., IRL Press,Oxford and Washington, 1985, or Wood, et al., PNAS, 82:1585-1588 (1985),both incorporated herein by reference.

In addition, claims are directed toward recombinant DNA vectorscomprising the claimed DNA molecules as well as bacterial cellscomprising such recombinant vectors. In a more particular embodiment,the bacterial cells are defined as E. coli.

The invention also includes polypeptide fragments of M. pneumoniaehaving M. pneumoniae P1 cytadhesin epitopes. More specific embodimentsare directed toward polypeptides further defined as being capable ofimmunospecifically binding to monoclonal antibody 6E7, ATCC# HB 8420.Similarly, an additional specific embodiment is directed towardspolypeptides defined as capable of immunospecifically binding tomonoclonal antibody 5B8, ATCC# HB-9586. Of course, where the recombinantpolypeptide is encoded by a DNA sequence inserted in a particular typeof expression vector, the polypeptide will be expressed as a fusionprotein. For example, when lambda gt11 is used as an expression vector,the M. pneumoniae polypeptide will be expressed in the form of abeta-galactosidase fusion protein. Although such fusion proteins areconsidered to be polypeptides and, thus, intended to be encompassed bythe claims of the present invention, one embodiment is specificallydirected to fusion proteins.

Additional claims are directed towards polypeptides comprising specificsequences as outlined in the claims. Of course, recombinant polypeptidesare included within the scope of the present invention. Moreover,synthetic polypeptides can be prepared from known amino acid sequences.The present invention is also meant to encompass any syntheticpolypeptide comprising the claimed amino acid sequences or polypeptideshaving conservative amino acid substitutions and essentially identicalfunction. Additional embodiments of the invention relate to vaccinescomprising such polypeptides and methods for inducing resistance to M.pneumoniae infection. Still further embodiments relate to diagnostickits comprising polypeptides having cytadhesin epitopes.

More specifically, the invention provides for a cytadhesin polypeptidecorresponding biologically to that produced by clones P1-7, P1-9 orP1-10, ATCC# 40386, 40385, or 40384, respectively. For the purposes ofthe present invention, a cytadhesin polypeptide correspondingbiologically to a polypeptide encoded by an identified recombinantvector is considered to be any polypeptide having similar or identicalfunction to that encoded by the specified recombinant vector. Suchpeptides may include synthetic peptides, including synthetic peptideshaving a slightly different amino acid sequence but essentially similarfunction. In a more specific embodiment, the polypeptides are furtherdefined as M. pneumoniae P1 polypeptides.

With even more particularity, the invention provides for a number of DNAmolecules comprising a recombinant DNA vector which includes therecombinant inserts of phages P1-7, P1-9, P1-10, ATCC# 40386, ATCC#40385, ATCC# 40384, respectively. The invention also includes arecombinant DNA vector which includes a recombinant insert of plasmidpMPN P1, ATCC# 67560. Bacterial strains comprising recombinant vectorswhich include such inserts are also included.

Finally, yet another feature of the present invention relates to amethod for screening mycoplasmal DNA for DNA sequences that correspondto those of M. pneumoniae P1, using the novel nucleotide sequences ofthe present invention. This method essentially comprises fractionatingmycoplasmal DNA to produce DNA fragments; separating the DNA fragmentsaccording to their sizes or molecular weights; hybridizing the DNAfragments with DNA molecules provided by the present invention; andidentifying at least one fragment which hybridizes to said DNA moleculesby means of a label.

Of course, the method will prove useful for isolation of M. pneumoniaeDNA sequences. However, it may also be useful for screening otherMycoplasmal species for homologous genes encoding M. pneumoniae P1. Forexample, the present inventors have observed that portions of the M.pneumoniae P1 gene are homologous to a gene from M. genitalium, and togenes from other mycoplasma, such as M. incognitus, and M.gallisepticum. Therefore, additional embodiments of the presentinvention relates to screening of those mycoplasmal species. Thespecificity of the novel nucleotides probes will depend, in part, on thehybridization conditions used. For example, where one desires to isolatenucleotide sequences encoding proteins homologous but not identical toM. pneumoniae P1, less stringent hybridization conditions should beused.

Methods which have proved particularly useful in fragmenting the DNAutilize restriction enzyme digestion or mechanical shearing. However,restriction enzyme digestion was utilized for the practice of thepresent invention. More particularly, the invention provides fordigestion of the mycoplasmal DNA with the restriction enzyme EcoRI.

The fragmented DNA can be separated into recognizable patterns usingvarious methods, the most useful of which take advantage of the varyingsizes of discrete DNA fragments. For example, DNA fragments can beseparated according to molecular weight by velocity sedimentationthrough a density gradient or, by molecular size exclusionchromatography. However, for purposes of the present invention, thepreferred technique is to separate the DNA fragments by electrophoresisthrough an agarose or polyacrylamide gel matrix.

The P1 hybridization probe can be conveniently labeled with radioactivenucleotides which allow for ready visualization of the hybridized DNA byautoradiography. Of course, other labeling techniques, including heavyisotopes or biotinylation, may also be used.

It should also be appreciated there is also no absolute requirement thatthe hybridization probes be derived from cloned M. pneumoniae P1 DNA.Since the present invention provides the complete gene sequence of M.pneumoniae P1, various oligonucleotide probes can be syntheticallyprepared on the basis of the disclosed sequence.

The substantially purified DNA molecules, recombinant DNA cloningvectors, recombinant cells, and recombinant proteins of the presentinvention may be used to prepare M. pneumoniae P1 polypeptide fragmentsor fusion proteins suitable for use as vaccines or reagents for use indiagnostic kits. Furthermore, the substantially purified DNA sequencesof the present invention are likely to prove useful as hybridizationprobes for selectively isolating mycoplasmal cytadhesin genes.Modification of the products of the present invention so as tofacilitate their utility in these or other areas is considered to bewell within its scope.

Characteristics of Deposited Microorganisms

Recombinant lambda gt11 vectors P1-7, P1-9, and P1-10 comprising clonesP1-7, P1-9, and P1-10 are available from the ATCC, accession # 40386,40385, and 40384, respectively. These clones comprise lambda gt11bacteriophages having a mycoplasmal DNA sequence ligated into the EcoRIsite within the beta-galactosidase gene.

E. coli HB101 comprising a recombinant pUC 19 plasmid vector having amycoplasmal DNA insert approximately 6 kbp in length ligated into theEcoRI site (plasmid pMPN P1) is available from ATCC, accession # 67560.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresisanalysis of protein samples during the purification of P1.

FIG. 1 (A) total protein extract from M. pneumoniae. Arrow indicates theposition of P1;

FIG. 1 (B) same sample from FIG. 1 (A) after a single passage throughthe anti-P1 affinity column;

FIG. 1 (C) protein eluted from the anti-P1 affinity column; and

FIG. 1 (D) P1 after preparative gel electrophoresis and electroelution.Proteins were separated by 7.5% polyacrylamide gel electrophoresis andstained with Coomassie blue. FIG. 2--The N-terminal 18 amino acidsequence of protein P1 and the 14-mer and 18-mer oligonucleotide probesdesigned to hybridize to the P1 gene. The 14-mer covers amino acids 1 to5 and the 18-mer covers amino acids 7 to 12. X═ACGT.

FIG. 3--M. pneumoniae DNA (12 ug/lane) digested with differentrestriction enzymes and separated by 0.7% agarose gel electrophoresis.

FIG. 3 (A) standard;

FIG. 3 (B) EcoRI;

FIG. 3 (C) Hae III;

FIG. 3 (D) Pst I;

FIG. 3 (E) Hind III;

FIG. 3 (F) BamHI;

FIG. 3 (G) Kpn I; and

FIG. 3 (H) Sal I.

FIG. 4--Southern blot analysis of M. pneumoniae genome. M. pneumoniaeDNA was digested with Hind III, separated by 0.7% agaroseelectrophoresis and transferred to nitrocellulose paper according to themethod of Southern (Mizusawa, et al., Nucleic Acids Res., 14:1319-1324(1986)). The nitrocellulose strip was then hybridized to the 14-mer (A)and 18-mer (B) probes labeled with ³² P. A single band (4.3 kb)hybridizes to both probes (arrow).

FIG. 5--Restriction enzyme map of the P1 gene. The first clone (62A)contains the 4.3 kb Hind III piece, and the second clone contains the 6kb EcoRI piece. Both the 14-mer and 18-mer probes hybridize to the DNAat a site very close to the first Sma I site. The cross-hatched boxrepresents the P1 structural gene.

FIG. 6--Complete nucleotide sequence and deduced amino acid sequence ofthe P1 gene. Both the coding and non-coding strand is shown. Thepresumed starting codon of P1 (ATG) is numbered as 1. In the 5' flankingregion, the possible promoter elements (-10 and -35) are underlined. The18 amino acids which match those determined by protein sequencing of P1are boxed (nucleotides 178-231). In the 3' flanking region, a sequencewith dyad symmetry, which may be a termination signal, is indicated bythe arrows and the "*" indicates mismatched sequences in this sequence.The complete P1 gene contains 4881 nucleotides coding for a protein of acalculated 176,288 daltons which includes an apparent leader peptide(see text).

FIG. 7--Plot of hydrophilicity value versus sequence position of P1according to the method of Hopp and Woods, Proc. Natl. Acad. Sci.,U.S.A., 78:3824-3828 (1981). Hydrophilicity values are averaged over sixamino acids through the length of P1; highest positive values representcharged hydrophilic regions.

FIG. 8--Location of the ten lambda gt11 clones within the P1 structuralgene. The predicted fusion protein size and DNA insert size of eachclone are given. Molecular weight values of the M. pneumoniae fusionproteins were calculated by subtracting the value of thebeta-galactosidase protein (116 kD). / indicates the location anddimension of the insert size. The numbers indicate nucleotidesencompassed by each clone. A "t" indicates that the clone extendsthrough the end of the P1 gene. As indicated in text, a TGA stop codonexists just downstream from the EV site.

FIG. 9--Gene sequence and deduced protein sequence of epitopes involvedin cytadherence by M. pneumoniae. The 13 amino acids within which oneepitope is located are underlined. Symbols corresponds to the following:,start of clone P1-7; , end of clone P1-7; *, start of clone P1-9; and,start of clone P1-10. The stop codon is indicated by the box.

FIG. 10--Hybridization of ³² P-labeled M. pneumoniae insert DNA fromclone P1-7 to M. pneumoniae genomic DNA digested with EcoRI (lane A),Hind III (lane B), Pst I (lane C), Sac I (land E), and Sma I (lane E).Molecular weights in kb are shown at the left.

FIG. 11--Immunoblot of cytadhesin fusion proteins using anti-P1 MAbs.Lane A represents total M. pneumoniae proteins reacted with a pool ofthe two MAbs designated 5B8 and 6E7 (see text). Lane B is thebeta-galactosidase protein reacted with a monoclonal Ab tobeta-galactosidase (Promega Biolab, Madison, Wis.). Lanes C and D areclones P1-7 and P1-9, respectively, reacted with MAb6E7. Lane E is cloneP1-10 reacted with MAb5B8.

FIG. 12--Immunophage blot of the ten different clones reacted with acute(I) and convalescent (II,III) sera of patients infected with M.pneumoniae. Numbers 7, 9, and 10 indicate clones P1-7, P1-9, and P1-10,respectively.

FIG. 13--Solubilized M. pneumoniae were run on a 7.5% gel prior totransfer to nitrocellulose for immunoblotting with hamster sera.Molecular weight standards corresponding to 25.7, 43.0, 68.0, 97.4, and200 kDa (bottom to top) are shown in Lane A. Lanes B (1:100) and C(1:1000) are dilutions of sera from an intra-nasally infected hamster.Lanes D (1:100) and E (1:1000) are dilutions of sera from a hamsterimmunized with the KLH-(P1) conjugate. Lane F was probed with normalhamster sera at a 1:100 dilution.

FIG. 14--Immunoblot of M. pneumoniae, M. genitalium, and M.gallisepticum proteins using M. pneumoniae anti-P1 rabbit monospecificAb. Similar results were obtained with M. genitalium anti-140 kDaantiserum. Molecular weight standards in kilodaltons are shown at theleft.

FIG. 15-Hybridization of the ³² P-labeled M. pneumoniae P1 gene to M.gallisepticum genomic DNA digested with BamHI (lane A); EcoRI (land B);HindIII (lane C); PstI (lane D). Identical patterns were observed usingthe M. genitalium 140 kDa gene. Molecular weight markers in kilobasesare shown at the left.

FIG. 16--Southern blot analysis of genomic DNA from M. hominis PG21, M.pulmonis, M. sualvi, M. fermentans K7, M. incognitus and M. fermentansPG18 digested with BamHI (B), EcoRI (E) and HindIII (H) and probed withthe ³² P-labeled P1 structural gene of M. pneumoniae. M. hominis, M.fermentans and M. incognitus are human pathogens: M. pulmonis is arodent pathogen; M. sualvi is a pig pathogen. Note the specifichybridization patterns indicating that P1 adhesin-related sequencesexist in each mycoplasma species.

FIG. 17--Southern blot analysis of genomic DNA from M. sualvi, M.fermentans K7, M. incognitus and M. fermentans PG18 digested with EcoRI(E) and HindIII (H) and probed with different subclones of the P1structural gene. Two groups of subclones were used: one group consistedof single copy regions G, L and M which correspond to nucleotides1771-2340, 4301-4338, 4339-4897, respectively and the other groupconsisted of multicopy regions B, C and D, which correspond tonucleotides -156-258, 259-909, and 910-1184, respectively. (See Infect.Immun. 56:3157-3161, 1988). The hybridization patterns exhibited by bothsets of probes were almost identical. This information further supportsthe direct sequence relationship between important regions of the P1adhesin gene of M. pneumoniae and analogue genes from "unrelated"pathogenic mycoplasmas.

FIG. 18--Restriction enzyme map of the P1 structural gene andsurrounding sequences. The boundary of each subclone from A to N ismarked. Restriction enzyme sites that cut more than once are numberedfrom the 5' end. Sau3A and TaqI cut many times in the P1 gene, but onlythe sites used for subcloning purposes are shown. Hatched bars indicatethe P1 structural gene. Numbers in parentheses indicate site numbers.

FIG. 19--Immunoblot of M. incognitus proteins using M. pneumoniaeanti-P1 and M. genitalium anti-140 rabbit monospecific antibodies.Several bands are immunoreactive. However, note the bands (arrow)identifying a common peptide of about 60 kDa. These data are consistentwith the hybridization profiles shown in FIGS. 1 and 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention relates to the isolation and nucleicacid sequence of the gene encoding the P1 cytadhesin protein from M.pneumoniae, the amino acid sequence of P1, and production of highlyantigenic P1 polypeptides, including fusion proteins. That aspect isdescribed in Section I below. A second aspect of the invention relatedto diagnostic and prophylactic methods and reagents employing nucleotidehomologies now discovered by the present inventors to have been highlyconserved among a wide variety of mycoplasmal species.

Section I

This aspect of the present invention is disclosed in terms of twogeneral approaches employed by the inventors to isolate clones andidentify nucleic acid sequences encoding M. pneumoniae P1 protein, orhighly antigenic M. pneumoniae polypeptides. The first general approachis primarily directed toward isolating, cloning, and sequencing thecomplete M. pneumoniae P1 gene, while the primary goal of the secondapproach is to identify particular nucleotide sequences encoding thefunctional cytadhesin domains of P1 and to produce antigenic cytadhesinpolypeptides suitable for use as diagnostic reagents or vaccines.

As indicated earlier, past attempts to clone the P1 gene were found tobe generally unsatisfactory. This failure was due, at least in part, tolack of a suitable method for unequivocally demonstrating that aparticular cloned DNA sequence actually represented the P1 gene.Fortunately, the present inventors have now discovered a techniqueallowing the complete structural P1 gene to be isolated and cloned. TheP1 gene has now been completely sequenced and the nucleotide sequenceunequivocally established as the structural P1 gene. In addition, theamino acid sequence of the complete P1 protein has been deduced from thenucleotide sequence.

Accordingly, the general approach described below represents aparticularly preferred approach for obtaining recombinant DNA clonescontaining the complete P1 gene. However, as illustrated below, themethod has also been successfully used for cloning partially complete P1genes.

The technique described below, disclosed for the first time by thepresent application, is one preferred method for obtaining recombinantDNA molecules and clones of the present invention. Of course, variationsof this method may also allow the gene to be cloned successfully. It isalso possible that other techniques could be successfully used to cloneM. pneumoniae P1. Any M. pneumoniae P1 gene cloned by such procedures isconsidered to be within the scope of the present invention, unless theclaims provide otherwise.

In general, recombinant clones produced in accordance with the presentinvention are made by first isolating mycoplasmal DNA. Any mycoplasmaencoding the P1 protein, may be used as a source of DNA. However,virulent strains of M. pneumoniae are preferred. These strains include,but are not limited to, M. pneumoniae isolated from infected humans oranimals, as well as defined strains maintained as laboratory cultures.The strain M. pneumoniae M129 is particularly preferred.

A number of methods for extracting DNA from prokaryotic organisms areknown which may, with possible routine modifications within ordinaryskill in the art, be used to extract mycoplasmal DNA. A preferred methodgenerally comprises lysing the organisms in a lysing buffer, forexample, sodium dodecyl sulfate in phosphate-buffered saline, extractingthe DNA from the lysed cell mixture with a suitable organic solvent suchas phenol, n-butanol or chloroform and reprecipitating the DNA with asuitable reagent such as ethanol, ethanol-acetate or isopropanol.

The extracted DNA is then fragmented. Any of a number of techniquessuitable for producing DNA fragments, such as mechanical shearing orpartial or complete restriction enzyme digestion, may be used. However,where a complete clone of the structural gene is desired, it isimportant to fragment the DNA so as to produce fragments at least about4.75-5.0 kb.

In general, digestion with restriction enzymes is a preferred method offragmenting the mycoplasmal DNA. Although any of a number of restrictionenzymes may be used (for example, see those listed in MOLECULAR CLONING,Maniatis, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., pp. 93-103), 1982 under properly selected digestion conditions,the present inventors have discovered that the M. pneumoniae genomecontains an EcoRI site on either side of the P1 structural gene, but notwithin the gene itself. Therefore, when EcoRI is used as a restrictionenzyme, complete digestion of M. pneumoniae DNA will produce a singleDNA fragment containing the entire structural gene. For this reason, itis especially preferred that the M. pneumoniae DNA be digested tocompletion with EcoRI in cases where a full length structural gene isdesired.

Conversely, if one desires to obtain a fragment containing only aportion of the P1 structural gene, complete digestion with Sal I, Pst,BamHI, Kpn I, Sma I, Sac I, Hind III, or Eco RV may be used since the P1structural gene is now shown to contain restriction sites for theseenzymes. Of course, it may be also possible to use these enzymes inpartial digestion procedures in cases where a full length P1 gene isdesired.

The fragmented DNA can be separated into a recognizable pattern usingvarious methods, the most useful of which take advantage of the varyingsizes of the discrete DNA fragments. For example, DNA fragments can beseparated according to molecular weight by velocity sedimentationthrough a density gradient, or by molecular size by gel exclusionchromatography. However, according to a technique preferred for thepurposes of the present invention, DNA fragments are separated byelectrophoresis through an agarose gel matrix and then transferred to anitrocellulose sheet so that an exact replica of the DNA fragments inthe gel is transferred to the nitrocellulose sheet.

A specific labelled probe is then applied to the nitrocellulose sheetunder selected hybridization conditions so as to hybridize withcomplementary DNA fragments localized on the sheet. Although variouslabels known to those of skill in the art may be employed, a radioactiveprobe is preferred. The sheet may then be analyzed by autoradiography tolocate particular fragments which hybridize to the probe.

Various hybridization probes may be used to detect the P1 structuralgene. For example, in experiments performed in conjunction withdevelopment of the present invention, the N-terminal amino acid sequenceof the P1 protein was determined and two oligonucleotides based on thissequence (the 14-mer and 18-mer probes described in Example I) wereutilized. In subsequent experiments, an EcoRI/Pst I restriction fragmentdigested from an incomplete P1 clone (62A) was used as a hybridizationprobe. However, as those of skill in the art will appreciate, any numberof suitable hybridization probes can now be prepared, based on sequencescontained within the complete P1 gene sequence provided for the firsttime by the present invention. All that is required is that the DNAfragments to be used as probes be of sufficient length to form a stableduplex or hybrid with the P1 DNA. Such fragments are said to be"hybridizable" in that they are capable of stable duplex formation.Generally, a DNA fragment of at least fourteen nucleotides in length (atetradecamer) is capable of forming a stable duplex. Of course, inpractice, it may be sometimes preferable to use a probe containing morenucleotides or alternatively, to use a battery of individual probes todetect a single fragment that hybridizes to each probe in the battery.

In any event, after DNA of the size range containing the desiredrestriction fragments is identified by the procedure generally describedabove, the fragment may be removed from the gel by a variety oftechniques known to those of ordinary skill in the art (e.g.,electroelution, dialysis, etc.) and cloned into an appropriate vector.Although it is likely that the restriction fragment containing the P1gene could successfully be cloned into several types of vectors, (e.g.,cosmids or phage), it is generally preferred to use a plasmid cloningvector, particularly where the desired restriction fragment is smallerthan 15 kbp.

After construction of recombinant vectors, the vectors are used totransform an appropriate host. In a preferred embodiment, the host is anE. coli cell of a type which is compatible with the selected vectortype. However, although the present invention is disclosed in terms ofE. coli host/vector systems, other host/vector systems are known in theart and may be employed where desired. For example, see those describedin DNA Cloning (Vol. II), P. M. Glover, ed., IRL Press, Oxford,Washington, D.C. (1985).

Transformation of host cells by the recombined vector is achieved usingstandard procedures known in the art. For example, where plasmid vectorsare employed, transformation is typically achieved by permeabilizingcompetent cells with calcium and contacting the permeabilized cells withthe recombinant vector DNA. Where bacteriophage vectors are employed,one may additionally choose to package the recombinant phage with phagecoat proteins, which affords direct transformation capability throughcell infection with a resultant increase in transformation efficiency.

Once the cells are successfully transformed with the recombinant vectorDNA, they are culture plated to provide individual recombinant clonalcolonies or plaques, a portion of which may express proteins or peptidesencoded by the M. pneumoniae P1 genome. In addition, clones may be usedas a source of M. pneumoniae DNA suitable for subcloning, sequencingstudies or use as hybridization probes.

The second general approach utilized by the present inventors relates tocloning and expression of M. pneumoniae DNA encoding polypeptides havinga cytadhesin epitope. The polypeptides so produced may be used asdiagnostic reagents or vaccines.

The focus of this approach differs somewhat from that described above inthat it is generally directed toward isolation and expression of M.pneumoniae DNA that encodes a particular functional domain of the P1protein, the domain responsible for cytadherence. In general then, thissecond approach involves fragmenting M. pneumoniae DNA by proceduressimilar to those described above and using the fragmented DNA toconstruct an M. pneumoniae DNA library or clone bank which is thenscreened with a reagent specific for clones encoding cytadhesinepitopes.

The DNA libraries may generally be constructed in either plasmids orbacteriophage, however, where expression of the cloned gene sequence isdesired, it is preferred that the library be constructed in anexpression vector. The lambda gt11 expression vector is particularlypreferred where expression of the cloned gene is desired because use oflambda gt11 has been found to ameliorate several problems generallyassociated with production of foreign proteins in E. coli. (See Huynh,et al., In DNA Cloning (Vol. I), E. M. Glover, ed., IRL Press, Oxford,Washington, D.C. (1985) and incorporated herein by reference.) Ofcourse, it is contemplated that a number of other vectors could also beused to generate and/or express the M. pneumoniae DNA library.

The library may be screened for clones containing the DNA sequencesencoding the cytadhesin domain of P1 by various procedures so long asthe screening reagents used allow isolation of a recombinant DNA cloneencoding at least a portion of the cytadhesin domain. For example, thepresent inventors used monoclonal antibodies previously shown torecognize the cytadhesin binding domain of M. pneumoniae P1 (SeeMorrison-Plummer, et al., Infect. Immun., 55:49-56 (1987)). Notably,those antibodies do not react with the DNA clones described by Trevino,et al.

Of course, since the present disclosure describes the nucleic acidsequence of the critical regions of the P1 gene, nucleic acidhybridization probes that selectively hybridize to these regions of theP1 genome may also be used for screening. (For examples of a nucleicacid screening procedure, see Huynh, et al., In DNA Cloning (Vol. I), E.M. Glover, ed., IRL Press, Oxford, Washington, D.C. (1985)). However,where one desires to screen with specific nucleic acid probes, lambdagt10 may be a preferred vector.

Once clones containing the M. pneumoniae cytadhesin epitopes areisolated, they may then be expanded and used as a source of M.pneumoniae DNA for sequencing studies. The sequence of the DNA insertsof the selected clones can then be compared with the complete DNAsequence of the P1 gene provided for the first time by the presentinvention. In this manner, the cloned inserts can be unequivocallyidentified as encoding all or part of the P1 protein.

DNA or deduced amino acid sequences from a battery of clones may then becorrelated with the antigenic phenotype of the polypeptides produced bysuch clones to precisely map the location of nucleotide sequencesencoding particular antigenic epitopes. Moreover, certain monoclonalantibodies specific for the P1 protein have been shown to inhibitcytadherence of M. pneumoniae and, therefore, are specific for thefunctional domain of P1 that mediates cytadherence. When thesemonoclonal antibodies are used for screening, the epitopes involved inmediating cytadherence can be mapped as well.

The recombinant DNA clones encoding all or part of the functional domainresponsible for cytadherence are particularly valuable. First, thepeptides expressed by such clones may be used as immunodiagnosticreagents to detect M. pneumoniae infection. More importantly, thepeptides may be incorporated into an antimycoplasmal vaccine. Inaddition, antigenic peptides comprising the cytadhesin specific epitopescan be synthesized, on the basis of the amino acid sequences deducedfrom the mapped nucleotide sequence and used as vaccines or antigens forimmunodiagnostic tests.

Finally, it should be pointed out that, for practical reasons, it mayoften be easier to demonstrate the P1 cytadhesin epitopes using amonoclonal antibody since polyclonal antiserum will usually containantibody molecules specific for regions of the P1 protein not associatedwith the cytadhesin domain as well as antibody molecules specific forcytadhesin epitopes. However, polyclonal antiserum capable of inhibitingP1 mediated cytadherence may also be used to demonstrate presence of thecytadhesin epitopes by a number of techniques generally known to thoseof skill in the art. For example, selected P1 polypeptides may be usedto extensively adsorb the polyclonal antiserum and adsorbed andnonadsorbed antiserum compared for the ability to inhibit cytadherence.By this procedure, specific polypeptides capable of significantlyreducing the antibody mediated inhibition of P1 mediated cytadherencemay be considered to express cytadhesin epitopes. In addition,cytadhesin epitopes may be demonstrated directly by their ability tocompetitively inhibit P1 mediated cytadherence in any of a number ofexperimental systems commonly used to measure cytadherence, described byMorrison-Plummer, et al., Infect. Immun., 53:398 (1986), or Krause andBaseman, Infect. Immun., 39:1180-1186 (1983).

Although the methodology described herein contains sufficient detail toenable one skilled in the art to practice the present invention, acommercially available technical manual entitled MOLECULAR CLONING(Maniatis, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) may provide additional details useful to assist practice of someaspects of the invention. Accordingly, this manual is incorporatedherein by reference.

The following examples are designed to illustrate certain aspects of thepresent invention. However, they should not be construed as limiting theclaims thereof.

EXAMPLE I

Isolation of a Recombinant Clone that Contains a DNA Sequence EncodingM. pneumoniae P1

This example is designed to illustrate the actual steps followed by theinventors in obtaining a specific recombinant clone that contained a DNAsequence encoding the mycoplasma P1 protein. However, this example isnot meant to represent the only procedure for cloning the P1 gene.

A. Culture of Mycoplasma and E. Coli

Virulent hemadsorbing Mycoplasma pneumoniae strain M129 in the sixteenthbroth passage was grown at 37° C. in 32 ounce glass prescription bottlescontaining 70 ml of Edward medium (Edward, J. Gen. Microbiol., 1:238-243(1947)). Glass adherent mycoplasmas were washed four times withphosphate buffered saline (PBS; pH 7.2) and collected by centrifugation(9,500×g 20 min.). Cells were harvested 72 hours after inoculation andstored at -70° C.

Escherichia coli strain HB101, DH5 alpha, and JM 107 were purchased fromcommercial sources and grown in LB broth (10 g/l Bacto-tryptone, 5 g/lBacto-yeast extract, 10 g/l NaCl, pH 7.5).

B. Purification of P1 Protein by Affinity Chromatography

The Pl protein was purified by antibody affinity chromatographyaccording to the method described by Leith and Baseman, J. Bacteriol.,157:678-680 (1984). Briefly, this method was as follows.

Four anti-P1 monoclonal antibodies secreted by hybridomas(Morrison-Plummer, et al., Infect. Immun., 55:49-56 (1987);Morrison-Plummer, et al., Infect. Immun., 53:398-403 (1986)) werecombined and purified by protein A-Sephadex column chromatography.Anti-P1 affinity columns were prepared by coupling 50 mg of purifiedanti-P1 antibody to 15 ml of cyanogen bromide activated Sephadex gel(Pharmacia, Piscataway, N.J.).

Pellets from 100 bottles of M. pneumoniae were suspended in 50 ml of 20mM Tris-HCl (pH 8.0), 0.2% sodium deoxycholate (Fisher Scientific), 0.1%sodium dodecyl sulfate (BDH Chemicals, Poole, England), 10 mM EDTA, and0.2% TRITON™-X-100 (octyl phenoxy polyethoxyethanol) containing 1 mMphenylmethylsulfonyl fluoride. Solubilization of proteins was assistedby passing the cell suspension through successively smaller gaugeneedles (22 to 27 gauge). Insoluble material was removed bycentrifugation at 100,000×g for 30 minutes.

Solubilized proteins were applied to the affinity column at 4° C. andwashed with 5 column volumes of the same buffer minus sodiumdeoxycholate. Bound protein was eluted with 0.1M acetic acid (pH 3)containing 0.15M NaCl and 0.1% SDS. The eluted protein was immediatelyneutralized with 1.0M Tris and concentrated in a pressureultrafiltration concentrator (Amicon, Danvers, Mass.).

As shown in FIG. 1 (panels A-C), this procedure selectively enriched forthe Mycoplasma pneumoniae cytadhesin protein P1 (165 kilodaltons).Approximately 400 ug of P1 protein was recovered after theimmunoaffinity step from an initial M. pneumoniae extract containing 300mg total protein.

As an additional purification step, the affinity column-purified P1 wasfurther processed by preparative gel electrophoresis through a 5%polyacrylamide-SDS gel. The gel was stained with Coomassie blue and theP1 protein band was cut out of the gel and electroeluted according tothe procedure of Hunkapiller, et al. (In Methods in Enzymology, C. H. W.Hirs and S. N. Timasheff (eds.) pp. 227-236 (1983)). About 60% recoverywas achieved after 24 hours of elution at room temperature in 50 mMammonium carbonate containing 0.1% SDS. The eluted protein was thenprecipitated in 80% methanol to remove SDS. SDS-PAGE analysis of therecovered P1 revealed that the sample contained intact P1 protein (FIG.1D), and the gel was deliberately overloaded to show the purity of thesample. Finally, the purified protein was shown to be P1 since itreacted with anti-P1 monoclonal antibodies in Western blot analyses(data not shown).

C. Determination of the N-Terminal Amino Acid Sequence of P1 Protein andPreparation of Specific Oligonucleotide Probes

The purified P1 protein was sequenced from the amino terminus with a gasphase protein sequencer. Approximately 50 ug of purified P1 was used(300 pmole) for each sequence analysis. Three separate determinationsyielded the sequence shown in FIG. 2.

The N-terminal amino sequence was used to deduce sequences foroligonucleotide probes. Two oligonucleotide probes complementary to allthe possible mRNA combinations encoding different portions of theprotein were synthesized, a 14-mer corresponding to amino acids 1-5 anda 18-mer corresponding to amino acids 7-12 (FIG. 2). The presentinventors used both C and T in the third position of the tryptophancodon of the 18 bp oligonucleotide in order to ensure hybridization withthe probe in the event that M. pneumoniae uses TGA (a stop codon inbacterial and eukaryotic systems) rather than TGG to encode tryptophan.The oligonucleotides were synthesized in the Department of Biochemistry,Baylor College of Medicine according to a procedure similar to thatdescribed by Alvarado-Urbina, et al., Science, 214:270-274 (1981),incorporated herein by reference, and purified by electrophoresis in 20%polyacrylamide gel containing 8M urea (Berent, et al., Biotech.,3:208-220 (1985)). For use as hybridization probes, the oligonucleotideswere labeled at the 5' end with Y-P³² -ATP by the T4-polynucleotidekinase reaction (Maniatis, et al., MOLECULAR CLONING, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982), pp. 122-127).

D. Southern Blot Analysis of M. pneumoniae DNA

M. pneumoniae DNA was prepared from exponentially growing cellsaccording to the following procedure. Pellets of M. pneumoniae weresuspended in 2.7 ml of PBS, lysed by the addition of 0.3 ml of 10%sodium dodecyl sulfate (SDS) and incubated with 10 ug of RNase for 30minutes at 37° C. Preparations were extracted three times with an equalvolume of redistilled phenol (equilibrated with 100 mM Tris [pH 8.0] -10mMEDTA [TE]) followed by dialysis overnight at 4° C. against a total of6 liters of sterile TE. Twelve ug of DNA was digested to completion withEcoRI, Hae III, Pst I, Hind III, BamHI, Kpn I or Sal I prior toelectrophoretic separation on 0.7% agarose gels. Gels were stained withethidium bromide and photographed under UV illumination (FIG. 3).

The gels were then analyzed according to the procedure of Southern, J.Mol. Biol., 98:503-519 (1975), incorporated herein by reference.Briefly, DNA was transferred to nitrocellulose filter paper with 20×SSC(0.3M sodium citrate, pH 7.0, 3M NaCl), rinsed once with 6×SSC, thenbaked at 80° C. for 2 hours under vacuum. Filters were prehybridizedovernight at 37° C. in 20 ml of prehybridization solution containing6×SSC, 60 mM sodium phosphate (pH 7.0), 5×Denhardt's solution (bovineserum albumin, polyvinylpyrrolidone, Ficoll at 1 mg/ml) and 0.1 mg/ml ofdenatured herring sperm DNA.

Hybridizations with the 14 base pair [bp] and 18 base pair [bp]oligonucleotide probes were carried out for 12 hours in 10 ml ofprehybridization solution plus 10% dextran sulfate and ³² P labeledoligonucleotide probes (3×10⁸ cpm) at 25° C. (14 bp, 14-mer) or 37° C.(18 bp, 18-mer). After incubation, filters were rinsed twice with 6×SSCat 4° C. (30 min. each), then washed twice in wash solution (3Mtetramethylammonium chloride, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1%SDS) at the appropriate temperature (14-mer at 37° C. and 18-mer at 45°C.) for 20 min. according to the procedure of Wood, et al., Proc. Nat.Aced. Sci., U.S.A., 82:1585-1588 (1985). After washing, filters wererinsed in 6×SSC at 4° C., dried and exposed to X-ray film using anintensifying screen.

Both probes hybridized to several DNA bands in each digestion, possiblybecause the probes were comprised of a mixture of oligonucleotidesformulated to react with all possible nucleotide sequences that couldencode the 12 N-terminal amino acids. A 4.3 kb Hind III fragmenthybridized most intensely to both the 14-mer and 18-mer (FIG. 4)strongly implicating this DNA fragment as containing the N-terminalsequence of P1.

E. Cloning DNA Fragments Encoding M. pneumoniae P1 Protein

To clone the DNA fragment described above, M. pneumoniae DNA wasdigested with Hind III, separated by agarose gel electrophoresis, andstained briefly with ethidium bromide. DNA in the 4.3 kb size range waseluted from the gel by electrophoresis onto DE-81 paper, eluted from thepaper with 20 mM Tris-HCl, pH 8.0, and 1.5M NaCl, then precipitated withethanol and redissolved in TE buffer.

The DNA was then ligated into the Hind III site of pUC 9. For thisprocedure, the plasmid was digested with an appropriate restrictionenzyme (Hind III) and the 5' end phosphate removed by calf intestinalalkaline phosphatase according to the procedure described on page 133 ofManiatis, et al., MOLECULAR CLONING, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1982). Mycoplasma DNA and vector were mixed at 1:1molar ratio and ligated at room temperature for 4 hours with T₄ DNAligase. After incubation, the reaction was stopped by adding EDTA to 10mM, diluted 5-fold with distilled H₂ O.

The ligated plasmid DNA was then used to transform competent HB101 orDH5 alpha E. coli cells according to the manufacturer's instructions(BRL, Bethesda, Md.). Transformants were selected on LB agar platescontaining 50 ug/ml of ampicillin. About 5,000 transformants wereobtained, of which 200 individual colonies were picked and grownovernight in 5 ml of LB broth containing 50 ug/ml of ampicillin. PlasmidDNA was isolated from overnight cultures by the alkaline lysis method(Ish-Horowicz and Burke, Nucleic Acid Res., 9:2989-2998 (1981)) andanalyzed on agarose gels.

To determine which insert-containing plasmids carried the P1 gene, DNAsfrom about 40 plasmids with inserts in the 4-5 kb range were blottedonto nitrocellulose filters. The filters were then hybridized to the ³²P labeled 14-mer and 18-mer oligonucleotide probes, washed and exposedto film as described above. Three clones hybridized strongly to bothprobes. By restriction endonuclease analysis the three clones containedthe same insert designated 62A (FIG. 5).

The DNA sequence which hybridized to both probes was narrowed to a 350bp Hae III restriction fragment by digesting the 62A plasmid with theHae III, separating the DNA on a 5% polyacrylamide gel, and transferringthe DNA from the gel onto nitrocellulose paper for hybridization witheach individual probe (data not shown). The 350 bp Hae III piece wassubcloned into M13mp18 and its sequence determined. It contains both the14-mer and 18-mer sequences, and most importantly the DNA has an openreading frame which codes for the 18 amino acids found by sequencing theamino terminus of the P1 protein (FIG. 6). Thus, clone 62A was shown tocontain at least a part of the structural gene encoding P1.

However, based upon the location of the sequenced Hae III fragment inthe 62A clone, the 4.3 kb Hind III DNA fragment was not large enough toencode the entire 165 kDa P1 protein. Therefore, an EcoRI/Pst Irestriction fragment from 62A was used to clone a larger DNA fragment.This procedure was performed as follows:

Plasmid 62A was isolated from overnight cultures by the alkaline lysismethod (Ish-Horowicz and Burke, Nucleic Acids Res., 9:2989-2998 (1981))and digested to completion with a mixture containing 500 units EcoRI and500 units Pst I. The resulting restriction fragment was purified byagarose gel electrophoresis, labeled by nick translation (Maniatis, etal., MOLECULAR CLONING, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982), pp. 109-112) and used to probe Southern blots of M.pneumoniae DNA digested to completion with EcoRI. This procedure wasperformed essentially as described above, except that the hybridizationconditions were more stringent including a higher temperature ofhybridization and wash (65° C.).

By this procedure, an M. pneumoniae DNA fragment approximately 6 kbp wasdetected. Accordingly, DNA in this size range was eluted from an agarosegel of the EcoRI-digested DNA by electrophoresis onto DE-81 paper,eluted from the paper with 20 mM Tris-HCl, pH 8.0, and 1.5M NaCl, thenprecipitated with ethanol and redissolved in TE buffer.

The DNA was then ligated into the EcoRI site of pUC 19, essentially asdescribed above and used to transform E. coli, as described above.Restriction enzyme analysis of the cloned insert indicated that the 6kbp insert overlapped clone 62A and was sufficiently large to encode theentire P1 protein. The restriction enzyme map depicting both the 4.3 kbpHind III fragment and the 6 kbp EcoRI fragment is shown in FIG. 5.

EXAMPLE II

Determination of the Complete DNA Sequence of the Gene EncodingMycoplasma pneumoniae P1 and Deduction of the Complete Amino AcidSequence of the P1 Protein

A. Sequencing of the P1 Gene

DNA sequences were determined by the dideoxy-chain-termination method ofSanger, et al., Proc. Natl. Acad. Sci., U.S.A., 74:5463-5467 (1977). M13sequencing kits were purchased from BRL and the reactions were performedaccording to the manufacturer's instructions except deoxy-7-deaza GTP(Boehringer Mannheim, Indianapolis, Ind.) was used in sequencingreactions in place of dGTP (Messing, et al., Nuc. Acid Res,, 9:309-321(1981)). Some DNA fragments were sequenced by subcloning appropriaterestriction enzyme fragments into an M13 phage vector (Messing, et al.,Nuc. Acids Res., 9:309-321 (1981)) and the single strand DNA purifiedfor use as a sequencing template. To sequence the rest of the P1 gene, alarge piece of DNA from the Pst I to the Sal I (see FIG. 5) wassubcloned into an M13 vector and a series of deletions from the 3' endwere generated by treating the double strand DNA with exonuclease IIIaccording to the method of Heinkoff, Gene, 28:351-359 (1981). Subcloneswith progressive deletions were selected for use as sequencingtemplates. Both strands of the entire P1 gene were sequenced. Nucleicacid and protein computer analyses were performed using the Microgenieprogram (Beckman, Palo Alto, Calif.). Comparisons of the P1 DNA anddeduced protein sequences were to the most recent releases of the NIHGenbank DNA sequence database and the National Biomedical ResearchFoundation protein sequence database, respectively.

B. Analysis of the P1 Nucleotide Sequence

The nucleotide sequence of the P1 gene is shown in FIG. 6. There is anopen reading frame of 4881 nucleotides and at the end of the gene is aTAG stop codon followed by 2 in-frame TAA stop codons 21 and 27 bpdownstream. This sequence could encode a protein of 1627 amino acidswith a calculated molecular weight of 176,288.

The nucleotide sequence includes a possible in frame translationinitiation site, ATG, 177 nucleotides from the P1 N-terminal sequence.There are conventional transcription initiation sites at -35 and -10upstream with a distance of 14 nucleotides between these two consensussequences (Reznikoff, et al., Ann, Rev, Genet., 19:355-387 (1985)), butno ribosomal binding site is observed between -10 and the initiationcodon. This predicts a protein with an extension of 59 amino acids fromthe N-terminus. Another possible translation initiation codon is the GTG(Gold, et al., Ann. Rev. Microbiol., 35:365-403 (1981)) at position 91.Use of this initiation site would predict a 28 amino acid precursor.

The open reading frame contains the 18 amino acids identified by gasphase sequencing (FIG. 6, Box). Comparison of the gas phase sequencewith the nucleotide sequence demonstrates that the inventors' hunch thatM. pneumoniae might use this codon to encode tryptophan was correct.

Moreover, it was observed that the 18 amino acids are found at position60-77 of the deduced protein instead of at the amino terminus of theopen reading frame. The reason for this apparent discrepancy could wellbe that P1, like many outer membrane proteins, is initially synthesizedas a precursor (Oliver, Ann. Rev. Microbial., 39:615-648 (1985)).Consistent with this hypothesis is the observation that the extra 59amino acids found at the amino terminus of the deduced protein appearlike a signal peptide; they include positively charged amino acidsfollowed by a stretch of hydrophobic amino acids (Oliver, Ann. Rev.Microbial., 39:615-648 (1985)). If protein P1 is indeed synthesized as aprecursor and processed into a mature protein, then the molecular weightof the mature protein would be 169,758 which is very close to the 165kDa reported earlier [Baseman, et al., J. Bacteriol., 151:1514-1522(1982); Krause, et al., Infect. Immun., 35:809-817 (1982); Leith andBaseman, J. Bacteriol., 157:678-680 (1984); and Morrison-Plummer, etal., Infect. Immun., 55:49-56 (1987)] and almost identical to the value(168 kDa) determined by Jacobs, et al., J. Clin. Microbiol., 23:517-522(1986) on SDS-PAGE.

Other relevant features of the sequence include a typical eubacterialpromoter (Reznikoff, et al., Ann. Rev. Genet., 19:355-387 (1985)) forRNA polymerase which is upstream of the first ATG codon, atapproximately -35 and -10. Also, a not-so-perfect invert repeat sequenceis detected 19 base pairs downstream from the TAG stop codon. Theinverted repeat sequence is a common feature of an RNA terminator(Rosenberg and Court, Ann. Rev. Genet., 13:319-353 (1979)). However, notypical ribosomal binding site is observed between -10 and theinitiation codon.

C. Determination of the Amino Acid Sequence of the P1 Protein

The complete amino acid sequence of the M. pneumoniae (FIG. 6) P1protein was predicted from the DNA sequence, also shown in FIG. 6. Thepredicted amino acid sequence is consistent with available informationabout protein P1: the predicted molecular weight of P1 approximates thereported values; and the predicted N-terminal amino acid sequence fitsexactly with the gas phase sequence analysis of purified P1 protein. Thepredicted P1 sequence contains more basic amino acids (Arg+Lys+His=169)than acidic (Asp=Glu=143) (isoelectric focusing data shows that P1 hasan isoelectric point at a basic pH). The predicted P1 contains nocysteine and thus has no intramolecular disulfide bonding, a findingwhich correlates with the previous observation that the P1 position inpolyacrylamide gels is not changed after exposure to sample buffercontaining reducing agents.

By referring again to FIG. 6, it can be seen that the predicted P1protein has several other interesting features: a) it contains highpercentages of hydroxy amino acids (17.7% are serine and threonine); andthe high proline content (13 of 26 amino acids) at the carboxy terminusis unusual and may place structural restraints on the protein and assistin regulating the topological organization of the cytadhesin in themembrane [Baseman, et al., J. Bacteriol, 151:1514-1522 (1982); Baseman,et al., In Molecular Basis of Oral Microbial Adhesion, S. E. Mergenhagenand B. Rosan (eds.), (1985); Kahane, et al., Infect. Immun., 49:457-458(1985); and Krause, et al., Infect. Immun., 35:809-817 (1982)].

It should be noted that FIG. 6 displays the actual nucleotide sequencedetermined by sequence analysis of the 6 kbp EcoRI fragment (plasmidpMPN P1) insert obtainable from ATCC# 67560. As those of skill in theart will appreciate, due to the redundancy of the genetic code, numerousother nucleotide sequences may be constructed which code for the sameamino acid sequence. Therefore, any nucleic acid sequence encoding forthe M. pneumoniae P1 protein as depicted in FIG. 6 is meant to beincluded within the scope of the present invention. This includesnucleotide sequences containing either the mycoplasmal (TGA) ortraditional (TGG) tryptophan codons.

D. Homology Between M. pneumoniae and Other Proteins Having Known AminoAcid Sequences

The deduced amino acid sequence for the P1 protein was compared to knownamino acid sequences listed in the National Biomedical ResearchFoundation protein sequence database. This analysis revealed that thepredicted P1 sequence is homologous to coat protein A of bacteriophageIke (protein P1 amino acid numbers 1308 through 1322 compared tobacteriophage amino acid numbers 240 through 254, 73.3% homology;257-290 vs. 231-264, 41.2% homology), protein 3A of Brome Mosaic virus(956-979 vs. 133-159, 52% homology), coat protein vp2 and vp3 of mousepolyomavirus (733-746 vs. 24-38, 66.7% homology), and coat protein Aprecursor of bacteriophage fd, M-13 and F1 (1296-1330 vs. 245-280, 51.3%homology). The 1290-1350 region of P1 also shares extensive homologywith cytoskeletal keratin of mammalian species. In addition, two regionsof P1 share extensive homology with human fibrinogen alpha chainprecursor (337-352 vs. 338-354, 70.6% homology; 822-852 vs. 544-565,59.1% homology). It is fascinating that parts of the P1 sequence arehomologous to specific viral coat proteins, mammalian cytoskeletalkeratin and to human fibrinogen alpha chain precursor. These findingsmay help explain observations of autoimmune-like mechanisms ofphysiopathology associated with mycoplasma disease (Biberfeld, S., Clin.Exp, Immunol., 8:319-333 (1971); Wise and Watson, Infect. Immun.,48:587-591 (1985)).

E. Analysis of Individual Antigenie Determinants Within the P1 Moleculeby Hydrophilicity Plotting

Cytadhesin P1 is strongly immunogenic and the appearance of anti-P1antibodies correlates with resolution of the atypical pneumoniae inducedby M. pneumoniae. Therefore, the recombinant P1 protein or selectedpeptides derived from the P1 protein provide attractive vaccinecandidates. The present inventors have performed experiments directedtowards mapping individual antigenic sites within the protein. Oneapproach is used to map the antigenic sites and is described below.

In general, antigenic sites are usually hydrophilic. Therefore, wherethe amino acid sequence of a protein is known, hydrophilicity plots maybe constructed which allow one to predict the location of antigenicdeterminants (Hopp and Woods, Proc. Natl. Acad. Sci., U.S.A.,78:3824-3828 (1981)). Hydrophilicity plotting of the predicted M.pneumoniae P1 sequence was performed using the Microgenie programobtained from Beckman (Palo Alto, Calif.). This analysis revealedpotential antigenic sites (FIG. 7) at positions 240-260, 280-304,314-333, 450-479, 680-690, 746-767, 898-913, 1244-1260, and 1476-1485.

EXAMPLE III

Expression of the Complete Recombinant P1 Protein

The following prophetic example is intended to describe methods by whichthe P1 gene could be expressed to provide a complete P1 protein.

The P1 protein could be expressed by ligating the piece of DNA thatincludes the first Hind III site through the second EcoRI site (see FIG.5) to a mycoplasma compatible vector, such as E. coli plasmid pAM120,then transforming fast growing mycoplasma species (such as Acholeplasma)for production of large quantities of P1. (See Dybvig, K., et al.,Science, 235:1392 (1987), which is incorporated herein by reference.)

The P1 gene could also be modified to express whole P1 in E. coli. Allthe UGA codons in the structural gene of P2 could be changed into UGG bysite specific mutagenesis. See Shortle, D., et al., Meth. in Enzymol.,100:457 (1983), which is incorporated herein by reference. Then apowerful E. coli promoter such as the lac promoter could be ligated tothe P1 gene to overproduce P1. Alternatively, an E. coli strain with UGAsuppressor phenotype (Raftery, L., et al., J. Bacteriol., 158:849(1984), which is incorporated herein by reference) could be used as hostto express the unmodified P1 gene.

Also, the P1 gene promoter is a unique mycoplasma promoter which can beused for the expression of other proteins in mycoplasma species.

EXAMPLE IV

Cloning, Sequencing, and Expression of Nucleotide Sequences Encoding theFunctional Cytadhesin Binding Domain of M. pneumoniae

This example describes the construction of the lambda gt11 recombinantDNA expression library of M. pneumoniae used to characterize the P1domain involved in cytadherence. In general, clones expressing P1epitopes were identified by screening the library with two anti-P1monoclonal antibodies known to block M. pneumoniae attachment toerythrocytes (RBCs) and respiratory epithelium.

A. Construction of the Lambda gt11 Library

1. Bacterial, Vector, and Restriction Enzymes

M. pneumoniae strain M129-B16 was cultured as described in Example I. E.coli Y1088 (American Type Culture Collection (ATCC#37195), Y1089(ATCC#37196), and Y1090 (ATCC#37197) were cultured in LB medium. Thesecell lines are available through the American Type Culture Collection orfrom Clontech Laboratories (Palo Alto, Calif.).

Lambda gt11 DNA arms and phage extracts were purchased from PromegaBiotech (Madison, Wis.). Enzymes used for constructing the genomiclibrary were from New England Biolabs (Beverly, Miss.); restrictionenzymes were from BRL (Gaitherburg, Md.).

2. Construction of the M. pneumoniae Genomic Library in Lambda gt11

M. pneumoniae strain M129-B16 genomic DNA library was constructed in theexpression vector lambda gt11 according to general procedures describedby Young and Davis, Proc. Natl. Acad. Sci., 80:1194-1198 (1983) andScience, 222:778-782 (1983) incorporated herein by reference.

More specifically, mycoplasmal DNA was extracted and fragmented asdescribed in Example I, but using mechanical shearing in place ofrestriction endonucleases.

The sheared DNA was then ligated to EcoRI linkers, and these DNAfragments were ligated into the EcoRI site in lambda gt11 armsessentially as described by Young and Davis (Proc. Natl. Acad. Sci.,80:1194 (1983) and Science, 222:778 (1983)). Briefly, this procedurecomprises incubating the vector DNA and the M. pneumoniae DNA fragmentsat high vector/insert ratio of 2:1 in ligation buffer (0.066M Tris-HCl,pH 7.5; 5 mMMgCl₂ ; 5mMDTT; 1 mM ATP) with 1U T4 DNA ligase at 12° C.for 2-16 hours.

Recombinant DNA was packaged to provide viable phage according toinstructions provided by the commercial supplier of the phage arms andphage extracts (Promega Biotech, Madison, Wis.). Alternatively,packaging extracts may be prepared and packaging reactions carried outaccording to protocols described on pages 256-268 of MOLECULAR CLONING.

The phage may then be titered by plating a small number of phage fromthe packaging mix (about 100) on E. coli Y1088 at 42° C., using 2.5 mlLB soft agar (pH 7.5) containing 40 ul of 40 mg/ml×gal and 40 ul of1MPTG for a 90 mm Petri dish. Plaques produced by the parental lambdagt11 phage are blue, while plaques produced by the recombinant phage arecolorless. (In a few cases, particular recombinant phage plaques willproduce a slight amount of blue color.)

The library may then be amplified by plating out the library at adensity of 10⁶ p.f.u. per 150 mm Petri dish, using 600 ul of Y1088plating cells per dish and fresh LB plates and incubating at 42° C.Plate stocks may be prepared as described by Davis, et al., BacterialGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1980).

Alternatively, it is possible to screen the lambda gt11 library withoutamplification. For this procedure, 0.1 ml Y1088 plating cells areinfected with ≦10⁵ plaque forming units at 37° C. for 15 minutes. Then0.5 ml of Y1090 plating cells and 7.5 ml LB soft agar are added. Themixture is poured into a two-day old 150 mm LB plate (pH 7.5).

B. Screening Lambda gt11 M. pneumoniae DNA Libraries With MonoclonalAntibody Probes

The M. pneumoniae DNA phage library was screened with a pool of twoanti-P1 monoclonal antibodies directed against unique M. pneumoniaeepitopes involved in cytadherence. The screening procedure was generallyperformed as follows.

E. coli Y1090 was grown to saturation in LB (pH 7.5) at 37° C. and 0.6ml of the Y1090 culture was mixed with up to 10⁵ p.f.u. in lambdadiluent for each plate. The phage were absorbed to the cells at 37° C.for 15 minutes. Then 7.5 ml of LB soft agar (pH 7.5) was added to theculture and the mixture was poured onto an LB plate (pH 7.5). The plateswere incubated at 42° C. for 3-4 hours and then placed at 37° C. Eachplate was then overlayed with a dry nitrocellulose filter disk which hadbeen saturated in 10 mM IPTG in water. The plates were then incubatedfor an additional 2-3 hours at 37° C. and removed to room temperature.The filters were then removed from the plate and the following stepswere performed.

First, the filters were rinsed briefly in TBS (50 mM Tris-HCl, pH 8.0,150 mM NaCl) and incubated in TBS plus 20% fetal calf serum for 15-30minutes. The filters were then incubated in TBS plus 20% fetal calfserum plus a mixture containing 1 ug/ml MAb6E7 and 2 ug/ml MAb5B8 forone hour. Preparation of these antibodies is described in Plummer, etal., Infect. Immune., 53:398-403 (1986), incorporated herein byreference.

The filters were then washed in TBS for 5-10 minutes, washed again inTBS plus 0.1% NONIDET™ P40 (NP-40; tert-octylphenoxypoly (ethoxyethanol)for 5-10 minutes. rewashed in TBS alone for 5-10 minutes, rinsed brieflyin TBS plus 20% fetal calf serum, and transferred to TBS plus 20% fetalcalf serum containing horseradish peroxidase-conjugated to goatanti-mouse immunoglobulin. The filters were then washed again in TBS,TBS plus 0.1% NONIDET™ P40(NP-40; tert-octylphenoxypoly (ethoxyethanol),and TBS. The filters were dried and 4-chloro-1-naphthol was used assubstrate to develop the immunoblots.

When the lambda gt11 M. pneumoniae genomic library was screened with thetwo monoclonal antibodies, ten independent clones that produced strongsignals were isolated. Eight of the clones reacted with both monoclonalantibodies, one clone (P1-7) reacted only with MAb6E7 and another clone(P1-10) reacted only with MAb5B8. The nucleotides encompassed by each ofthese clones is indicated by FIG. 8.

C. Analysis of the Recombinant Phage Clones

The following experiments were performed in order to furthercharacterize the mycoplasmal proteins produced by the recombinant phage.Positive signal-producing phage were grown in E. coli Y1090 as describedin MOLECULAR CLONING, pp. 64-65. DNA was extracted by a rapidsmall-scale plate lysate method using 2 units of EcoRI to excise the M.pneumoniae DNA inserts essentially as described in MOLECULAR CLONING,pp. 371-372.

1. Seguencing of the M. pneumoniae DNA Inserts

DNA sequences of the recombinant phage inserts were determinedessentially as described in Example II. The results of this analysis areshown in FIGS. 8 and 9. By comparing the sequences of these clones tothe complete P1 gene sequence (FIG. 6), the cytadhesin binding domain ofthe M. pneumoniae P1 protein was mapped to the C-terminal region of theP1 gene. The sequences of three clones were of particular utility infurther mapping antigenic epitopes of P1. These clones were P1-7, P1-9,and P-10. As shown in FIG. 9, clone P1-7 starts at position 4067 andends at position 4185; clone P1-9 starts at position 4148 and extendsbeyond the end of the P1 gene. These two clones both contain nucleotides4148-4185. These nucleotides code for a P1 polypeptide thirteen aminoacids in length, the thirteen amino acids that contain the epitopereactive with the cytadherence-blocking MAb6E7. Clone P1-10 starts atposition 4202 and extends beyond the P1 gene. This clone is nonreactivewith MAb6E7, yet shares a stretch of nucleotides that overlap with cloneP1-9; further demarcating the thirteen amino acid cytadherence relatedepitope.

Therefore, a key domain of adhesin P1 that mediates the cytadherence ofvirulent M. pneumoniae to respiratory epithelium has been mapped to athirteen amino acid region located in the C-terminal end of the P1molecule. In addition, the present studies have established that asecond cytadhesin epitope, recognized by MAb5B8, is C-terminal toposition 4202. Therefore, the C-terminal end of the P1 protein appearsto be the primary effector region of the P1 molecule. It is interestingthat the carboxy terminus of the P1 protein is proline rich (13 of thelast 26 amino acids are proline). This hydrophobic domain may functionto anchor the carboxy terminal end of the P1 molecule in the M.pneumoniae membrane.

2. The Thirteen Amino Acid Cytadhesin Epitope is Unique to M. pneumoniaeP1

By comparing the sequence of the P1-7 probe to the known DNA sequence ofthe complete P1 gene, it was determined that the P1 molecule containedonly one copy of the thirteen amino acid epitope described above.However, in order to determine whether or not this epitope was unique toM. pneumoniae, the following experiment was performed.

Mycoplasma DNA was digested with different restriction enzymes (BamHI,EcoRI, Hind III, Pst I, Sac I, Sma I) and fractionated by agarose gelelectrophoresis, essentially as described in Example ID above. However,the DNA insert from clone P1-7 was used as a hybridization probe.Hybridization was carried out at 68° C. overnight according to Maniatis,et al., MOLECULAR CLONING, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982), pp. 382-289. The results of this procedure, shownin FIG. 10, demonstrate that the cytadherence related epitope of cloneP1-7 occurs only once in the M. pneumoniae genome.

D. Analysis of M. pneumoniae P1 Cytadhesin Peptides

The following studies were undertaken to further characterize thecytadhesin polypeptides produced by the recombinant lambda gt11bacteriophage.

It will be appreciated by those familiar with the lambda gt11 fusionsystem, that the site used for insertion of foreign DNA is a uniqueEcoRI cleavage site located within the lacZ gene, 53 base pairs upstreamfrom the beta-galactosidase translation termination codon. Because thesite of insertion for foreign DNA in lambda gt11 is within thestructural gene for beta-galactosidase, foreign DNA sequences in thisvector have the potential to be expressed as fusion proteins withbeta-galactosidase. The position within the beta-galactosidase genechosen for fusion with foreign DNA sequences, corresponds to a regionnear the carboxy terminus of the beta-galactosidase protein.

Fusion proteins expressed by the recombinant clones of the presentinvention were analyzed by Western blotting. This procedure wasperformed essentially as follows. M. pneumoniae protein (2 mg) wassuspended in 0.3 ml of PBS, and an equal volume of 100 mM Tris (pH 6.8)-2% S.D.S. -20% glycerol -2% 2-mercaptoethanol-0.02% bromophenol bluebuffer (SP buffer) was added. Samples were boiled for 5 minutes.Recombinant fusion proteins were harvested from plate lysates ofindividual clones by scraping soft agarose overlays from the plates,passing them through a 22 gauge needle into a Corex tube and eludingwith 4m of SM buffer for two hours at 4° C. The agarose was pelleted bycentrifugation at 10,000×g for 15 minutes at 4° C. prior totrichloracetic acid precipitation of the supernatant by the addition ofcold trichloracetic acid, for a final concentration of 10%. Samples wereincubated at 4° C. overnight prior to centrifugation at 10,000×g for 20minutes at 4° C. Supernatants were discarded, and pellets were washedtwice with 1 ml of PBS, suspended in 200 ul of SP buffer, andneutralized with 1 ul of 5N NaOH. Samples were boiled for 5 minutes andsolubilized proteins were electrophoresed on a 5.0% polyacrylamide gelprior to electrophoretic transfer to nitrocellulose paper (Towbin, etal., Proc. Natl. Acad. Sci., U.S.A., 76:4350-4354 (1979)).

After protein transfer, the nitrocellulose was cut into strips andreacted with a pool of the two MAbs (monoclonal antibodies) designated5B8 and 6E7. For this procedure, nitrocellulose blots were blocked in1.5% bovine serum albumin (BSA)+1.5% gelatin in TBS for 3-4 hours priorto incubation with the pooled monoclonal antibodies. The finalconcentration of the antibodies in the reaction mixture was 2 ug/ml 5B8and 1 ug/ml 6E7 in a buffer comprising TBS plus 20% FCS. Blots wereincubated with the diluted antibody preparation overnight at roomtemperature with shaking, following by three ten minute washes with TBS.Horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:2000 inTBS containing 0.75% BSA -0.75% gelatin was added to the blots andincubated with shaking for 3-4 hours at room temperature. Blots werewashed three times for ten minute periods with TBS prior to substratedevelopment.

The results of this procedure, shown in FIG. 11, show the representativeclones produced fusion proteins larger than the control lambda gt11beta-galactosidase protein. However, except for clone P1-7, the size ofeach fusion protein was much smaller than that predicted from the sizeof the corresponding recombinant DNA insert. This finding may beexplained as resulting from early termination of the cytadhesin peptidedue to the presence of the TGA codon at position 4556.

The present inventors have discovered that M. pneumoniae utilizes thiscodon for tryptophan, while E. coli reads UGA as stop signal. Therefore,when E. coli is used as a host for a vector containing the recombinantPneumoniae insert, a prematurely truncated polypeptide may be produced.

E. Cytadhesin Peptides Can Be Used For Serodiagnosis of M. pneumoniaeInfection

Studies have shown that adhesin P1 is highly immunogenic (Hu, et al.,Science, 216:313-315 (1982)) and patients infected with M. pneumoniaeexhibit neutralizing antibodies to the P1 adhesin (Leith, et al., J.Exp. Med., 157:502-516 (1983)). Since the isolated clones express P1cytadhesin peptides, these clones were analyzed for reactivity with seraof patients with early and late stages of M. pneumoniae infection.Normal human sera was used as a control. These experiments wereperformed by the immunophage blot method. Briefly, this procedure wasperformed as follows. Individual recombinant phages were dotted on alawn of E. coli Y1090. The plates were incubated at 42° C. for 3-5hours. Then a nitrocellulose filter (HAHY, M) previously saturated with10 mM IPTG was overlayed on individual plates and incubation continuedat 37° C. overnight. Filters were removed and reacted with sera from M.pneumoniae infected patients or normal human controls essentially asdescribed in FIG. 12 using horseradish peroxidase-conjugated goatanti-human immunoglobulin, and 4-chloro-1-naphthol to develop theimmunoblots.

The results of this procedure, shown in FIG. 12, indicated that fusionproteins produced by all ten anti-P1 MAb reactive clones also reactedwith acute and convalescent sera of M. pneumoniae infected patients butdid not react with normal human serum. Therefore, the cytadherencerelated P1 peptides or fusion proteins described herein may be used forserodiagnosis of patients infected with M. pneumonias.

F. Preparation of Recombinant Antigens from the Lambda gt11 RecombinantClones

It is often useful to have preparative amounts of polypeptides specifiedby a cloned piece of DNA. For some purposes, for instance,radioimmunoassays, it is sufficient to have a crude E. coli lysatecontaining an antigen specified by the cloned DNA of interest. Thisprophetic example illustrates how a crude lysate containing a cytadhesinpeptide fusion protein can be prepared by expressing a lambda gt11recombinant as a lysogen in E. coli 1089 (E. coli Delta lac U169proA+Delta lon ara D139 strA hsl A150 [chr::Tn10] (p MC9)). Therecombinant fusion protein would be produced by lysogenizing Y1089 withthe lambda gt11 clone of interest. The lysogen would be grown to highcell density, lacZ-directed fusion protein production induced by theaddition of IPTG to the medium, and the cells harvested and lysed.

More specifically, the Y1089 cells would be grown to saturation in LBmedium (pH 7.5/0.2% maltose) at 37° C. and then infected with theselected lambda gt11 recombinant phage (preferably P1-7) at amultiplicity of approximately 5 for 20 minutes at 32° C. in LB medium(pH 7.5) supplemented with 10 mM MgCl₂. The cells would then be platedon LB plate at a density of approximately 200/plate and incubated at 32°C. At this temperature, the temperature sensitive phage repressor isfunctional. Single colonies would be tested for temperature sensitivityat 42° C. by spotting cells from single colonies using steriletoothpicks onto two LB plates. The first plate would be incubated at 42°C. and the second at 32° C. Clones growing at 32° C. but not at 42° C.are assumed to be lysogens. Lysogens should arise at a frequency between10% and 70%.

The crude lysate would then be prepared from the lambda gt11 recombinantlysogen by incubating 100 ml of LB medium with a single colony of theY1089 recombinant lysogen at 32° C. with aeration. When the culture hasgrown to an optical density of 0.5 measured at 600 mm, the temperatureof the culture would be increased to 42°-54° C. as rapidly as possibleand the culture incubated at the elevated temperature for 20 minuteswith good aeration. IPTG would be added to 10 mM and the culture isincubated at 37°-38° C. for approximately one hour. At this stage, theY1089 lysogen will sometimes lyse, even though the Y1089 does notsuppress the mutation, causing defective lyses (S100) in lambda gt11.The reason for this is that the S100 amber mutation is leaky and foreignproteins accumulating in E. coli often render it susceptible to lysis.Therefore, the longest incubation time achievable at 37°-38° C. withoutlysis occurring should be determined for each individual recombinantlysogen. After incubation, the cells would be harvested in a Beckman J.A.-ten rotor at 5,000 r.p.m. for 5 minutes 27°-37° C. The cells wouldthen be rapidly resuspended in 1/20 to 1/50 of the original culturevolume in a buffer suitable for protein and the resuspended cells arerapidly frozen in liquid nitrogen. When the frozen cells are thawed,essentially complete lysis of the induced lysogen results.

If crude antigen is required, the crude lysate described above could beused. However, if pure antigen is needed, the beta-galactosidase fusionprotein would be purified by any of a number of methods known to thoseof skill in the art. The most rapid method of purification takesadvantage of the size of the beta-galactosidase fusion protein(approximately 114 kDa). Since only a few proteins in E. coli are largerthan beta-galactosidase, the fusion protein is often resolved from otherproteins on SDS-polyacrylamide gels. Preparative gels could be used toisolate large quantities of denatured protein. If pure antigen in nativeform is required, then the fusion protein could be prepared by classicalcolumn chromatography.

G. Synthesis of a Synthetic Peptide Containing the Amino Acid CytadhesinEpitopes

The following prophetic example describes methods for preparingsynthetic polypeptides containing cytadhesin epitopes. M. pneumoniae P1polypeptides could be prepared by any of a number of methods known tothose of skill in the art. These methods include but are not limited tosolid and liquid phase chemical synthesis and biological in vitrosynthesis. For example, see Marglin and Merrifield, Annu. Red. Biochem.,39:841-866 (1970); Merrifield, et al., Biochemistry, 21:5020-5031(1982); Pelham and Jackson, Eru. J. Biochem., 67:247-256 (1976); andShinnick, et al., Ann. Rev. Microbiol., 37:425-446 (1983), allincorporated herein by reference. Of course, where an mRNA translationsystem is used, e.g., reticulocyte lysate system, it is important toprepare mRNA from the DNA clones of the present invention.

Techniques for preparing the mRNA from DNA clones are known in the art.For example, see those described in Chapter 2, 1987 Promega BiologicalResearch Products Catalogue, obtainable from Promega Biolabs, 2800 SouthFish Hatchery Road, Madison, Wis. 53711-5305 and incorporated herein byreference. A preferred method for preparing a synthetic peptide may befound in U.S. Pat. No. 4,493,795 issued to Nestor, Jr., et al., andincorporated herein by reference. A second method is found in U.S. Pat.No. 4,474,757, issued to Arman, et al., and also incorporated herein byreference.

H. Preparation of M. pneumonias Compositions for use as M. pneumoniasVaccines

Of course, it is also likely that the cytadhesin peptides may beeffectively used as vaccines to prevent atypical pneumonias caused by M.pneumonias. The preparation of vaccines which contain peptide sequencesas active ingredients is generally well understood in the art, asexemplified by U.S. Pat. Nos.: 4,474,757; 4,493,795; 4,608,251;4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, allincorporated herein by reference.

This prophetic example describes preparation and administration of suchvaccines. In general, immunogenic compositions suitable foradministration as vaccines could be formulated to include one or more ofthe antigenic epitopes produced by the recombinant cells of the presentinvention or synthetically prepared. The antigens could be included inoptimal amounts, for example, approximately equimolar or equi-antigenicamounts. Typically, such vaccines are prepared as injectables: either asliquid solutions or suspensions, solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation could also be emulsified. The reactive immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine could contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccine.

In addition, immunogenicity of cytadhesin peptides could be increased byconjugation of a carrier molecule, for example, dipalmityl lysine. (SeeHopp, Mol. Immunol., 21:13-16 (1984) incorporated herein by reference.)

The proteins or polypeptides could be formulated into the vaccine asneutral or salt forms and administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective and immunogenic. The vaccines could be conventionallyadministered parenterally, by injection, for example, eithersubcutaneously or intramuscularly. Additional formulations which aresuitable for other modes of administration might include oral orintranasal formulations. The quantity to be administered will depend onthe subject to be treated, capacity of the immune system to synthesizeantibodies, and the degree of protection desired. Precise amounts ofactive ingredient required to be administered will depend on thejudgment of the practitioner and may be peculiar to each individual.However, suitable dosage ranges will be on the order of 1 to 100 ugactive ingredient per individual. Suitable regimes for initialadministration and booster shots will also be variable, but may betypified by an initial administration followed by subsequentinoculations or other administrations.

In many instances, it may be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the antigens as described below.

I. Use of Synthetic P1 Epitope Peptide for Vaccine to Block Cytadherenceof M. pneumoniae to In Vitro Targets

A 13 amino acid peptide fragment of P1 having the sequence Gly - Ile -Val - Arg - Thr - Pro - Leu - Ala - Glu - Leu - Leu - Asp - Gly waschosen for pilot vaccine studies in hamsters. Hamsters were a convenientmodel to use for M. pneumoniae infection because of their susceptibilityto mycoplasma infection. As described above, this 13 amino acid sequenceincludes an epitope that reacts with anti-P1 monoclonal antibodies whichspecifically block cytadherence of viable M. pneumoniae to in vitrotargets. (See Example IV., C.).

The rationale was to use a synthetic peptide that mimics the biologicalfunction of attachment of pathogenic mycoplasmas. This peptide/vaccineshould elicit an immune response which would interferes withadhesin-receptor interactions.

The peptide was synthesized commercially (Peninsula Laboratories,Belmont, Calif.), and 10 mg of synthetic peptide was next coupled to 5mg keyhole lympet hemocyanin (KLH, Sigma, St. Louis, Mo.) usingestablished procedures (H. L. Niman et al., Proc. Natl. Acad. Sci.U.S.A. 82:7924-7928 (1985)).

In order to determine whether the synthetic peptides still possessed animmunological conformation similar to native P1 protein, the peptide wasbound to microELISA polyvinylstyrene wells and tested in an ELISA priorto and post conjugation to KLH, essentially as described byMorrison-Plummer et al., Infection and Immunity 53(2):398-403 (1986)).

Two monoclonal antibodies were used in the ELISA: (1) H13.6E7: anantibody raised against intact M. pneumoniae, and shown to bind to anepitope within the 13 amino acid sequence and to block cytadherence ofM. pneumoniae to in vitro targets (J. Morrison-Plummer et al., Infectionand Immunity 53(2):398-403 (1986)); and (2) H12.5B8: an antibody of thesame isotype as H13.6E7. H12.5B8 binds to an epitope of P1 protein notrepresented in the synthetic 13 amino acid peptide. H12.5B8 also blockscytadherence, however. (J. Morrison-Plummer et al., Infection andImmunity 53(2):398-403 (1986)). As shown in Table 1, H13.6E7 bound thesynthetic peptide with and without conjugation to KLH. Monoclonalantibody H12.5B8 did not bind the protein before or after conjugation.Immunogenicity of the synthetic peptide post conjugation to KLH appearedto remain intact as evidenced by the binding of Mab H13.6E7.

The inventors next evaluated the response of human acute/convalescentsera from M. pneumoniae-infected patients using the same peptide-KLHconjugate as the antigen in the ELISA. Sera were diluted 1:100 andreacted with 200 ng of the conjugated peptide. Two to three foldincreases in IgG titer of the convalescent compared to acute sera wereobserved. Values ranged from 0.057 to 0.079 for acute and from 0.135 and0.254 for convalescent sera. Normal (uninfected sera from 5 individualstested at the same dilution) gave values of <0.078.

To determine whether the hamster would recognize the KLH-(P1) peptide asimmunogenic, hamsters were then immunized with the conjugated KLH-(P1)synthetic peptide. These experiments were also designed to determinewhether the immune response would elicit high titered antibodies capableof blocking attachment of M. pneumoniae to eucaryotic host cells.

Six hamsters were inoculated (intraperitoneally and subcutaneously) withthe KLH-(P1) peptide conjugate. Each hamster received two injections of200 μg total protein prior to analysis of the sera by immunoblot usingwhole solubilized M. pneumoniae. For that procedure, solubilized M.pneumoniae were run on a 7.5% gel prior to transfer to nitrocellulosefor immunoblotting with hamster sera. FIG. 13 demonstrates that thepeptide elicited production of antibodies that reacted with solubilizedM. pneumoniae, i.e., elicited an immune response. As shown in FIG. 13,lanes B and C, the hamsters inoculated with the conjugate developedantibodies directed against the P1 protein of M. pneumoniae. Normalhamster sera had no detectable response at 1:100 dilution (lane F).

                  TABLE 1                                                         ______________________________________                                        Antigenicity of the synthetic peptide                                         before and after conjugation to KLH.                                          ELISA REACTIVITY.sup.1                                                        MONOCLONAL                                                                    KLH                    SYNTHETIC PEPTIDE.sup.3                                ANTIBODY.sup.4                                                                            M. pneumoniae.sup.2                                                                      without KLH Peptide-                                   ______________________________________                                        H13.6E7    ≧1.500                                                                             .434        .986                                       H12.5B8    ≧1.500                                                                             .077        .128                                       ______________________________________                                         .sup.1 Values shown represent absorbance readings at 410 nm. Standard         deviations were <20% of the mean. Maximum value is 1.5.                       .sup.2 M. pneumoniae were air dried onto microELISA wells at a                concentration of 2 ug per well ().                                            .sup.3 The synthetic peptide P1 was dissolved in 0.1M acetic acid and         diluted to 200 nm per well in pH 8.6 coating buffer.                          .sup.4 H13.6#7 recognizes a cytadherencerelated epitope in P1. H12.5B8 is     of the same isotype but recognizes a different cytadherencerelated epitop     in P1.                                                                   

Having demonstrated that the synthetic peptide conjugate was immunogenicin the hamster model, the inventors examined whether the antibodiesgenerated were capable of blocking attachment of M. pneumoniae to invitro targets, an attractive attribute for a synthetic peptide vaccine.Erythrocytes were used as targets in this study because of thesimilarity of the receptor sites to those of respiratory epithelium(78). As shown in Table 2, immune hamster sera effectively blocked M.pneumoniae attachment to sheep red blood cells. Normal hamster sera hadno blocking antibodies.

                  TABLE 2                                                         ______________________________________                                        Effects of sera from KLH-synthetic peptide                                    immunized hamsters on cytadherence                                            of M. pneumoniae to in vitro targets                                          SERA.sup.b            MYCOPLASMA Attachment                                   INHIBITION.sup.d                                                                        DILUTION    CPM.sup.c   %                                           ______________________________________                                        NHS       1:10        21,821       0                                          NHS       1:50        20,690       0                                          LEP       1:10         6,119      69                                          LEP       1:50        12,795      35                                          REP       1:10         3,206      84                                          REP       1:50        14,933      24                                          ______________________________________                                         .sup.a Attachment of viable mycoplasmas was assessed using previously         published methods (69)                                                        .sup.b Sera consisted of normal hamster sera (NHS), and two representativ     sera from hamsters immunized with KLHP1-peptide conjugate as described in     the text; left ear punch (LEP) and right ear punch (REP).                     .sup.c Counts per minute (CPM), are shown as the mean of triplicate           samples.                                                                      .sup.d Percent inhibition represents control cpm (NHS) minus the mean cpm     of test samples divided by the mean cpm of controls without antibody          × 100.                                                             

These studies demonstrate the efficacy of the peptide in evoking aneffective immune response in actual animal models.

J. Immunoassay For M. pneumoniae Antibodies

As demonstrated by Example IV E., certain of the P1 polypeptides areknown to react with antisera from patients infected with M. pneumoniae.Accordingly, these polypeptides may be used as antigens in immunoassayprocedures. These assays are well known to those of skill in the art.For examples of such assays, see Nisonoff, Introduction to MolecularImmunology, 2nd Ed., Sinaues Associates, Inc., Sunderland, Mass. (1984)and U.S. Pat. No. 4,376,110, both incorporated herein by reference.

The following prophetic example is designed to illustrate suchprocedures. Generally, for detection of antibody in aqueous samples, theantigen, or antigen composition, is preferably adsorbed, or otherwiseattached, to an appropriate adsorption matrix, for example, the insidesurface of a microtiter dish well, and an aqueous suspectedantibody-containing composition contacted therewith to causeimmunocomplex formation. The matrix is then washed to removenon-specifically bound material and the amount of material which isspecifically immunocomplexed thereto determined, typically through theuse of an appropriate labeled ligand.

The cytadhesin polypeptides provided by the present invention may alsobe incorporated into a diagnostic kit. Such kits are widely used inclinical settings because they often offer greater convenience andsimplicity than other assays. A number of kits might be utilized in thepractice of the present invention, for example, a kit comprising acarrier compartmentalized to receive at least one, at least two, or atleast three or more containers and to maintain said containers enclosedconfinement.

A first container might include one or more of the M. pneumoniaeantigens, or antigen-containing compositions. Alternatively, or inaddition, the kits will include antibody compositions having specificityfor one or more of the antigens. Both antibody and antigen preparationsshould preferably be provided in a suitable titrated form, with antigenconcentrations and/or antibody titers given for easy reference inquantitative applications.

The kits will also typically include an immunodetection reagent or labelfor the detection of specific immunoreaction between the providedantigen and/or antibody, as the case may be, and the diagnostic sample.Suitable detection reagents are well known in the art as exemplified byradioactive, enzymatic or otherwise chromogenic ligands, which aretypically employed in association with the antigen and/or antibody, orin association with a second antibody having specificity for the antigenor first antibody. Thus, the reaction is detected or quantified by meansof detecting or quantifying the label. Immunodetection reagents andprocesses suitable for application in connection with the novelcompositions of the present invention are generally well known in theart.

Section II: Cross-Hybridization Between the Cytadhesin Genes in VariousMycoplasmal Species

This section is designed to illustrate the cross-hybridization betweenthe cytadhesin genes of a wide variety of mycoplasmal species.

EXAMPLE V

Cross-Hybridization Between the Cytadhesin Genes of Mycoplasmapneumoniae and Mycoplasma genitalium and Genomic DNA of Mycoplasmagallisepticum

This example is designed to illustrate the cross-hybridization betweenthe cytadhesin genes of M. pneumoniae and M. genitalium and genomic DNAof M. gallisepticum. The inventors utilized several methodologies todemonstrate cytadhesin-related sequences in M. gallisepticum.

A. Culture of M. pneumoniae, M. genitalium, and M. gallisepticum

Virulent M. pneumoniae strain M129-B16 and M. gallisepticum strain (S6)were grown in 32 oz. (ca. 950 ml) prescription bottles in 70 ml ofmodified Edward medium at 37° C. for 72 hr. [D. G. Edward, J. Gen.Microbial. 1:238-243 (1947)]. Glass-adherent M. pneumoniae organismswere washed four times with phosphate-buffered saline (PBS; pH 7.2) andcollected by centrifugation (9,5000×g, 20 min). With M. gallisepticum,which adheres less avidly to glass, both glass-adherent and detachedorganisms were combined and washed by centrifugation. Mycoplasmagenitalium G37 was grown in SP-4 medium as described by Tully, et al.and similar procedures were employed for M. pneumoniae (J. Infect. Dis.,139:478-482 (1979); Science, 195:892-894 (1971)).

B. Demonstration of Cross-reactive Epitopes Shared Between M.genitalium, M. pneumoniae, and M. gallisepticum

Total protein immunoreactivity of M. gallisepticum, M. genitalium and M.pneumoniae was determined by solubilizing mycoplasma pellets, separatingproteins using 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis and electrophoretically transferring the proteins tonitrocellulose paper prior to immunoblotting with rabbit monospecificand mouse monoclonal anti-adhesin antibodies [J. Morrison-Plummer etal., Isr. J. Med. Sci. 23:453-457 (1987)].

FIG. 14 illustrates an immunoblot of M. pneumoniae, M. genitalium, andM. gallipepticum proteins using M. pneumoniae anti-P1 rabbitmonospecific Ab. Cross reactive epitopes shared by the P1 protein of M.pneumoniae, the 140 kDa adhesin protein of M. genitalium and a 155 kDaprotein of M. gallisepticum were observed (FIG. 14) using rabbitmonospecific antibody to the P1 and 140 kDa proteins of M. pneumoniaeand M. genitalium, respectively. Similar results were obtained with M.genitalium anti-140 kDa antiserum.

Blots of these three mycoplasma strains were also reacted withcytadherence-blocking monoclonal antibodies (mAbs) generated against theP1 and 140 kDa proteins. Monoclonal antibodies to the P1 protein of M.pneumoniae exhibited strong reactivity against the homologous P1 andweak reactivity against the 140 kDa protein of M. genitalium [J.Morrison-Plummer et al., Isr. J. Med. Sci. 23:453-457 (1987)]. Likewise,mAbs to the 140 kDa protein of M. genitalium were strongly reactive inthe homologous blot and weakly reactive against the P1 protein of M.pneumoniae. Neither group of mAbs exhibited reactivity against M.gallisepticum suggesting the absence of related epitopes.

C. Demonstration of Cross-reactivity Between M. genitalium, M.pneumoniae, and Genomic DNA of M. gallisepticum

This example describes the results of studies analyzing hybridizationbetween P1 and 140 kDa genes and M. gallisepticum genomic DNA.

M. gallisepticum cells were resuspended in 2.7 ml of PBS buffer, lysedby addition of 0.3 ml of 10% SDS and incubated with 50 μg RNase(Boehringer Mannheim Biochemicals, IN) per ml for 30 min at 37° C. DNApreparations were extracted three times with an equal volume of phenolequilibrated with 1M Tris (pH 8.0), once with phenol:chloroform (1:1),and once with chloroform: isoamy1 alcohol (24:1). Sodium acetate (3N;0.1volume) was added to the DNA preparation, and the DNA was precipitatedwith ethanol.

Southern blots of M. gallisepticum DNA digested with differentrestriction enzymes were hybridized with a ³² P-labeled P1 5.6 Kb Eco RIfragment or a gene encoding the 140 kDa structural genes of M.genitalium essentially as described by S. F. Dallo et al., Infect.Immun. 57:1059-1065 (1989). FIG. 15 describes the hybridization of the³² P-labeled M. pneumoniae P1 gene to M. gallisepticum genomic DNAdigested with BamHI (lane A); EcoRI (land B); HindIII (lane C); PstI(lane D). Hybridizations were performed under low stringency conditions[J. G. Tully et al., Science 195:892-894 (1971)]. Using the M.genitalium 140 kDa gene, both M. pneumoniae and M. genitalium cytadhesingenes displayed the same patterns of hybridization with genomic DNA ofM. gallisepticum. This is the first demonstration of a genetic basis fora family of adhesin-related genes among mycoplasmas pathogenic for manand animals (FIG. 15).

D. Direct Structure--Function Relationship Between These MycoplasmaAdhesins

To further establish a direct structure-function relationship betweenthese mycoplasma adhesins, M. gallisepticum genomic DNA was probed withthe ³² P-radiolabeled subclone of the P1 structural gene that encodesthe 13 amino acid epitope mediating M. pneumoniae cytadherence (Gly -Ile - Val - Arg - Thr - Pro - Leu - Ala - Glu - Leu - Leu - Asp - Gly).This subclone hybridizes to the M. genitalium 140 kDa gene under lowstringency conditions. No hybridization was detected with M.gallisepticum DNA, suggesting that this specific region of thestructural gene of M. gallisepticum differs considerably from M.pneumoniae and M. genitalium. These data are consistent with the lack ofimmunoreactivity using cytadherence-blocking mAbs to the P1 and 140 kDaadhesin proteins as described above.

EXAMPLE VI

Cross-Hybridization Between Cytadhesin Genes of Diverse PathogeneticMycoplasmas Representing a Family of Adhesin-Related Molecules

This example is designed to illustrate the cross-hybridization betweendiverse pathogenic mycoplasmas representing a family of adhesin-relatedmolecules. As demonstrated above, the P1 and 140 kDa adhesin-relatedmolecules of M. pneumoniae show high gene and protein sequencehomologies with M. gallisepticum and M. genitalium. The inventorsexpanded on this information and discovered a family of adhesin-relatedsequences among many pathegenic mycoplasmas.

A. Southern Blot Analysis of Genomic DNA from Several Different Sourcesof Mycoplasmas

FIG. 16 demonstrates southern blot analysis of genomic DNA from M.hominis PG21, M. pulmonis, M. sualvi, M. fermentans K7, M. fermentansPG18, and M. incognitus using ³² P-labeled P1 structural gene of M.pneumoniae, M. hominis as a radioactive probe. M. fermentans (K7 andPG18 are different human isolates) and M. incognitus are humanpathogens; M. pulmonis is a rodent pathogen; M. sualvi is a pigpathogen. Each of the genomic DNAs were digested with BamHI (B), EcoRI(E) and HindIII (H).

The results of this southern blot analysis demonstrates specifichybridization patterns shared among the various species of mycoplasmasindicating that P1 adhesin-related sequences exist in each of themycoplasma species tested.

B. Southern Blot Analysis of Genomic DNA from Several Different Sourcesof Mycoplasmas Using Different Subclones of P1 Structural Gene as theProbe

A southern blot analysis of genomic DNA from M. sualvi, M. fermentansK7, M. incognitus and M. fermentans PG18 probed with different subclonesof the P1 structural gene is shown in FIG. 17. The genomic DNA wasdigested with EcoRI (E) and HindIII (H). Two groups of P1 structuralsubclones were used: one group consisted of single copy regions G, L andM, which correspond to nucleotides 1771-2340, 4301-4338, 4339-4897,respectively (also shown by letter in FIG. 18) and the other groupconsisted of multicopy regions B, C and D, which correspondnucleotides--156-258, 259-909, and 910-1184, respectively. (See Su, etal., Infect. Immun. 56(12):3157-3161, 1988, incorporated herein byreference).

The restriction enzyme map of the P1 structural gene and surroundingsequences is presented in FIG. 18. The boundary of each subclone from Ato N is marked. Restriction enzyme sites that cut more than once arenumbered from the 5' end. Sau3A and TaqI cut many times in the P1 gene,but only the sites used for subcloning purposes are shown. Hatched barsindicate the P1 structural gene. Numbers in parentheses indicate sitenumbers.

Probe subclone B is restriction site EcoRI(site 1) to SmaI, 414 basepairs, located from nucleotides -156 to 258. Probe subclone C isrestriction site Sma(1) to PstI, 651 base pairs, located fromnucleotides 259 to 909. Probe subclone D is restriction site Pst(1) toBamHI, 275 base pairs, located from nucleotides 910 to 1184. (See FIG.18).

Probe subclone G is restriction site KpnI(site 1) to Sau3A (internal),570 base pairs, located from nucleotides 1771-2340. Probe subclone L isrestriction site SalI to EcoRV, 236 base pairs, located from nucleotides4103-4338. Probe subclone M is restriction site EcoRV to TaqI(site 1),559 base pairs, located from nucleotides 4339 to 4897. (See FIG. 18).

The hybridization patterns exhibited by both sets of probes were nearlyidentical. Using probe G, L, and M, consistent bands with molecularweights of approximately 4 kb and 1.8 kb (EcoRi) and 4 kb and 2-3 kb(HindIII) were observed in the two human pathogens, M. fermentans, M.incognitus. This information further supports the direct sequencerelationship between important regions of the P1 adhesin gene of M.pneumoniae and analogue genes from unrelated pathogenic mycoplasmas.

C. Immunoblot Analysis of M. incognitus Proteins using Anti-P1 M.pneumoniae and M. genitalium anti-140 kD Antibodies

FIG. 19 demonstrates the immunoreactive bands in human pathogen, M.incognitus proteins using M. pneumoniae anti-P1 and M. genitaliumanti-140 kD rabbit monospecific antibodies. From this analysis, severalbands are found to be immunoreactive. Note the bands indicated by thearrow, identifying a common immunoreactive peptide of approximately 60kDa identified with both antibodies. These data are consistent with thehybridization profiles shown in FIGS. 16 and 17. E. Conclusions

P1 adhesin of M. pneumoniae has been shown to be an essential virulencefactor because it mediates cytadherence and its absence results innon-cytadherence and avirulence. P1 adhesin, and related sequencesincluding P30 adhesin of M. pneumoniae, represent a family ofadhesin-related and virulence related molecules found in diversepathogenic mycoplasmas. These mycoplasmas normally exhibit littlegenomic homologies. Example V presenting data on the cross-hybridizationwith the poultry pathogen, M. gallisepticum, reinforces this correlationamong many pathogenic mycoplasmas and that these molecules may serve asimportant diagnostic and vaccine reagents.

The foregoing description has been directed to particular embodiments ofthe invention in accordance with the requirements of the Patent Statutesfor the purposes of illustration and explanation. It will be apparent,however, to those skilled in this art, that many modifications andchanges in the apparatus and procedure set forth will be possiblewithout departing from the scope and spirit of the invention. It isintended that the following claims be interpreted to embrace all suchmodifications and changes.

What is claimed is:
 1. A method of detecting mycoplasmal DNA in a biological sample comprising the steps of:(a) obtaining a biological sample suspected of Mycoplasma contamination or infection, wherein said sample is selected from the group consisting of blood, cell culture or tissue; (b) isolating DNA from said biological sample; (c) hybridizing said DNA with a labelled polynucleotide segment encoding a portion of M. pneumoniae P1 polypeptide, wherein said polynucleotide segment is selected from a group consisting of polynucleotide segment -156 to 258, polynucleotide segment 259 to 909, polynucleotide segment 910 to 1184, polynucleotide segment 1771 to 2340, polynucleotide segment 4103 to 4338, and polynucleotide segment 4339 to 4897 of the nucleotide sequences shown in FIG. 6, said polynucleotide segment is capable of hybridizing under moderate stringency hybridization conditions to mycoplasmal DNA present in at least two of the following, M. genitallure, M. gallisepticum, M. fermentans, M. incognitus, M. hominis, M. pulmonis, and M. sualvi, wherein the homology required for moderate stringency hybridization is at least approximately 75% hornology between the labelled polynucleotide segment and the DNA in the biological sample; and (c) identifying DNA which hybridizes to said labelled polynucleotide segment by means of detecting said hybridization.
 2. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. fermentans.
 3. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. pulmonis.
 4. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. gallisepticum.
 5. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. incognitus.
 6. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. hominis.
 7. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. sualvi.
 8. The method of claim 1 wherein said mycoplasmal DNA is DNA from M. genitaIium.
 9. The method of claim 1 wherein a mixture of said polynucleotide segments is used in said hybridization step (c).
 10. A method for detecting mycoplasmal DNA in a biological sample comprising of the following steps:(a) obtaining a biological sample suspected of Mycoplasma contamination or infection, wherein said sample is selected from the group consisting of blood, cell culture or tissue; (b) isolating DNA from said biological sample; (c) hybridizing said DNA with labelled polynucleotide segment 4147-4185 of the nucleotide sequences shown in FIG. 6, which encodes the following amino acid sequence: Gly - Ile -Val - Arg - Thr- Pro - Leu- Ala - Glu - Leu - Leu - Asp - Gly, under moderate stringency hybridization conditions, wherein the homology required for moderate stringency hybridization is at least approximately 75% homology between the labelled polynucleotide segment and the DNA in the biological sample; and (d) identifying DNA which hybridizes to said labeled polynucleotide segment by means of detecting said hybridization.
 11. The method of claim 10 wherein a mixture comprising polynucleotide segment -156 to 258, polynucleotide segment 259 to 909, and polynucleotide segment 910 to 1184 of the nucleotide sequences shown in FIG. 6 is used to detect mycoplasmal DNA in a biological sample.
 12. The method of claim 10 wherein a mixture comprising polynucleotide segment 1771 to 2340, polynucleotide segment 4103 to 4338, and polynucleotide segment 4339 to 4897 of the nucleotide sequences shown in FIG. 6 is used to detect mycoplasmal DNA in a biological sample. 